Binding fusion proteins, binding fusion protein-drug conjugates, XTEN-drug conjugates and methods of making and using same

ABSTRACT

The present invention relates to binding fusion protein compositions comprising targeting moieties linked to extended recombinant polypeptide (XTEN), binding fusion protein-drug conjugate compositions, and XTEN-drug conjugate compositions, isolated nucleic acids encoding the compositions and vectors and host cells containing the same, and methods of using such compositions in treatment of diseases, disorders, and conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/977,035, filed Dec. 21, 2015, which is a continuation applicationof U.S. application Ser. No. 13/631,361, filed Sep. 28, 2012 which is acontinuation application of International Patent ApplicationPCT/US2011/030992, filed Apr. 1, 2011 which claims the benefit of U.S.Provisional Application Ser. Nos. 61/341,720 filed Apr. 2, 2010, and61/341,996 filed Apr. 8, 2010, each of which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under SBIR grant2R44GM079873-02 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 27, 2012, isnamed 32808731.txt and is 2,247,117 bytes in size.

BACKGROUND OF THE INVENTION

Antibodies or immunoglobulins are molecules that recognize and bind tospecific cognate antigens or ligands. Because of their exclusivespecificities, antibodies, particularly monoclonal antibodies, have beenwidely used in the diagnosis and treatment of a variety of humandiseases.

Full-length antibodies comprise two heavy chains linked together bydisulfide bonds and two light chains, each light chain being linked toone of the heavy chains by a disulfide bond. Each chain has a variabledomain (V_(H) or V_(L)) at the N-terminus and one or more constantdomains at the C-terminus; the constant domain of the light chain isaligned with and disulfide bonded to the first constant domain of theheavy chain, and the light chain variable domain is aligned with thevariable domain of the heavy chain. Each of the variable domains of theheavy and light chain includes framework regions (FRs) and hypervariableregions and an intrachain disulfide bond. (See e.g. Chothia et al., J.Mol. Biol. 186:651-663 (1985); Novotny and Haber, Proc. Natl. Acad. Sci.USA 82:45924596 (1985); Padlar et al., Mol. Immunol., 23(9): 951-960(1986); and S. Miller, J. Mol. Biol., 216:965-973 (1990). Antibodies canbe derived from native antibodies or synthesized that may includecombinations of heavy and light chain variable domains so as to form anantigen binding site. The types of antibody fragments include, forexample, Fab, Fab′, F(ab′)₂, Fv, scFv, Fd, and Fd′ fragments. However,expression of antibody fragments in bacterial hosts, including domainantibody fragments (dAb), Fv fragments, single-chain Fv fragments(scFv), Fab fragments, Fab′2 fragments, and many non-antibody proteins(such as FnIII domains) can result in the formation of inclusion bodiesin the cytoplasm, adding to the complexity and cost of production (Kou,G., et al., 2007, Protein Expr Purif. 52, 131; Cao, P., et al. 2006,Appl Microbiol Biotechnol., 73, 151; Chen, L. H et al., 2006, ProteinExpr Purif.; 46, 495). In addition, the stability and/or productionyields of scFv or Fab fragments of natural antibodies produced in hostcells have been found to be insufficient. Honneger et al., J. Mol.Biol., 309:687-699 (2001), and the stability of scFv fragments is notalways correlated with expression yield in the bacterial periplasm (Wornet al., J. Mol. Biol., 305:989-1010 (2001). The many factors that affectthe periplasmic expression yield and/or stability of scFv are not yetfully understood and may be unpredictable. In addition, the proceduresfor extracting periplasmic proteins are not as robust as extraction fromthe cytoplasm, which contributes to low yields. Thus, because thereremains a need for improving the process of producing antibodies andantibody fragments, particularly in soluble form, finding alternativeproteins that can bind antigens and that can be produced with improvedyields in cell culture, especially with a bacterial cell culture, isdesirable.

SUMMARY OF THE INVENTION

The present invention relates generally to novel, selectable bindingfusion proteins useful as agents for the treatment of any disease orcondition that is improved, ameliorated, or inhibited by theadministration of proteins that bind certain proteins, carbohydrates orglycoprotein targets associated with the disease, disorder or condition.In particular, the present invention provides compositions of bindingfusion proteins comprising extended recombinant polypeptides with anon-repetitive sequence and unstructured conformation (XTEN) linked toone or more polypeptide targeting moieties exhibiting binding affinityto certain targets. The binding fusion proteins of the embodimentsdisclosed herein exhibit one or more or any combination of theproperties and/or the embodiments as detailed herein.

In some embodiments, the invention provides isolated binding fusionproteins comprising an extended recombinant polypeptide (XTEN) linked toa targeting moiety with binding affinity to a target selected from Table1 or Table 2, wherein the fusion protein exhibits a terminal half-lifethat is longer than about 48 h, or about 72 h, or about 96 h, or about120 h, or about 10 days, or about 21 days, or about 30 days whenadministered to a subject. In one embodiment of the foregoing, the XTENis characterized in that the sequence comprises at least about 36, or atleast about 72, or at least about 98, or at least about 144, or at leastabout 288, or at least about 576, or at least about 864, or at leastabout 1000, or at least about 1400, or at least about 2000, to about3000 amino acid residues, the sum of glycine (G), alanine (A), serine(S), threonine (T), glutamate (E) and proline (P) residues constitutesmore than about 80%, or about 85%, or about 90%, or about 95%, or about96%, or about 97%, or about 98%, or about 99%, or about 100%, of thetotal amino acid sequence of the XTEN, the XTEN sequence issubstantially non-repetitive in that (i) the XTEN sequence contains nothree contiguous amino acids that are identical unless the amino acidsare serine, (ii) at least about 80% of the XTEN sequence consists ofnon-overlapping sequence motifs, each of the sequence motifs comprisingabout 9 to about 14 amino acid residues, wherein any two contiguousamino acid residues does not occur more than twice in each of thesequence motifs; or (iii) the XTEN sequence has an average subsequencescore of less than 3, the XTEN sequence lacks a predicted T-cell epitopewhen analyzed by TEPITOPE algorithm, wherein the TEPITOPE algorithmprediction for epitopes within the XTEN sequence is based on a score of−6, −7, or −8, or −9, or −10, the XTEN sequence has greater than 90%random coil formation, or about 95%, or about 96%, or about 97%, orabout 98%, or about 99% random coil formation as determined by GORalgorithm; and the XTEN sequence has less than 2% alpha helices and 2%beta-sheets as determined by Chou-Fasman algorithm. In anotherembodiment of the foregoing, the XTEN is further characterized in thatthe sum of asparagine and glutamine residues is less than 10% of thetotal amino acid sequence of the XTEN, the sum of methionine andtryptophan residues is less than 2% of the total amino acid sequence ofthe XTEN

In another embodiment, the XTEN of the binding fusion protein is furthercharacterized in that no one type of amino acid constitutes more thanabout 16%, or 24%, or about 30% of the XTEN sequence. In anotherembodiment, the XTEN of the binding fusion protein is furthercharacterized in that at least about 80%, or at least about 90%, or atleast about 91%, or at least about 92%, or at least about 93%, or atleast about 94%, or at least about 95%, or at least about 96%, or atleast about 97%, or at least about 98%, or at least about 99% of theXTEN sequence consists of non-overlapping sequence motifs wherein eachof the sequence motifs has about 12 amino acid residues and wherein thesequence of any two contiguous amino acid residues does not occur morethan twice in each of the sequence motifs, and the sequence motifsconsist of four to six types of amino acids selected from glycine (G),alanine (A), serine (S), threonine (T), glutamate (E) and proline (P).In one embodiment, greater than 90% of the XTEN sequence consists ofnon-overlapping sequence motifs, wherein the sequence motifs are fromone or more sequences of Table 3. In another embodiment, the XTENsequence exhibits at least about 80%, or at least about 90%, or at leastabout 91%, or at least about 92%, or at least about 93%, or at leastabout 94%, or at least about 95%, or at least about 96%, or at leastabout 97%, or at least about 98%, or at least about 99% or 100% sequenceidentity to a sequence selected from any one of Table 4, Table 11, Table12, Table 13, Table 14, or Table 15, when optimally aligned. In anotherembodiment, the invention provides an isolated binding fusion proteincomprising a sequence that has at least about 80%, or at least about90%, or at least about 91%, or at least about 92%, or at least about93%, or at least about 94%, or at least about 95%, or at least about96%, or at least about 97%, or at least about 98%, or at least about 99%or 100% sequence identity to a sequence selected from any one of Table25, Table 40 or Table 41.

The subject binding fusion proteins exhibit enhanced pharmacokineticproperties. In one embodiment the enhanced pharmacokinetic property is aterminal half-life that is greater than about 24 h, or greater thanabout 48 h, or greater than about 72 h, or greater than about 96 h, orgreater than about 120 h, or greater than about 144 h, or greater thanabout 7 days, or greater than about 10 days, or greater than about 14days, or greater than about 21 days when administered to a subject,wherein the pharmacokinetic properties are ascertained by measuringblood concentrations of the fusion protein over time afteradministration of a dose to a subject. In one embodiment, the enhancedpharmacokinetic property encompasses an increase in terminal half-lifeof at least about two fold, or at least about three-fold, or at leastabout four-fold, or at least about five-fold, or at least aboutsix-fold, or at least about eight-fold, or at least about ten-fold, orat least about 20-fold compared to the targeting moiety not linked tothe XTEN and administered to a subject at a comparable dose.

In one embodiment, the targeting moiety of the isolated binding fusionprotein is selected from the group consisting of antibody, antibodyfragment, scFv, diabody, domain antibody, cytokine receptor, andimmunoglobulin superfamily receptor. In one embodiment, the targetingmoiety is a scFv. In another embodiment, the targeting moiety is a scFvwith binding affinity to Her2. In some embodiments, the binding fusionprotein is multivalent, comprising two, or three, or four, or five, orsix, or seven, or eight targeting moieties. In one embodiment of theforegoing, the multivalent targeting moiety is a scFv. In one embodimentthe multivalent targeting moieties can exhibit specific binding affinityto the same target, wherein the targets are selected from Table 1 orTable 2. In one embodiment, the multivalent targeting moieties canexhibit specific binding affinity to two or more targets, wherein thetargets are selected from Table 1 or Table 2. In any of the embodimentsdescribed in this paragraph, the binding affinity constant (K_(d)) forthe one or more targeting moieties of the subject binding fusion proteinand a target ligand is less than about 10⁻⁴ M, alternatively less thanabout 10⁻⁵M, alternatively less than about 10⁻⁶M, alternatively lessthan about 10⁻⁷M, alternatively less than about 10⁻⁸M, alternativelyless than about 10⁻⁹M, or less than about 10⁻¹⁰ M, or less than about10⁻¹¹ M, or less than about 10⁻¹²M.

In one embodiment, the binding fusion protein can comprise a second XTENhaving at least about 48 amino acid residues linked to the N-terminus ofthe binding fusion protein, wherein the expression of the binding fusionprotein in a host cell comprising an expression vector coding thebinding fusion protein is enhanced compared to the expression in a hostcell comprising an expression vector encoding a corresponding bindingfusion protein lacking the second XTEN. In one embodiment of theforegoing, the second XTEN exhibits at least 90% sequence identity, orat least about 91%, or at least about 92%, or at least about 93%, or atleast about 94%, or at least about 95%, or at least about 96%, or atleast about 97%, or at least about 98%, or at least about 99% or 100%sequence identity to an XTEN sequence selected from AE48, AM 48, AE624,AE912 or AM923. In another embodiment of the foregoing, theincorporation into a host cell of a polynucleotide encoding a bindingfusion protein comprising N-terminal XTEN can result in an expressionlevel that is enhanced at least 50%, or at least about 75%, or at leastabout 100%, or at least about 150%, or at least about 200% or morecompared to the expression levels in a comparable host cell with apolynucleotide encoding a binding fusion protein without the N-terminalXTEN.

The invention provides binding fusion proteins in variousconfigurations. In one embodiment, the invention provides an isolatedbinding fusion protein of formula I:(XTEN)_(x)-TM-(XTEN)_(y)  Iwherein independently for each occurrence, XTEN is an extendedrecombinant polypeptide comprising greater than about 36 to about 3000amino acids with a substantially non-repetitive sequence wherein the sumof glycine (G), alanine (A), serine (S), threonine (T), glutamate (E)and proline (P) residues constitutes more than about 80% of the totalamino acid sequence of the XTEN, x is either 0 or 1, y is either 0 or 1,wherein x+y≥1, and TM is a targeting moiety with specific bindingaffinity to a target selected from Table 1 or Table 2.

In another embodiment, the invention provides an isolated binding fusionprotein of formula II:(XTEN)_(x)-TM1-L-TM2-(XTEN)_(y)  IIwherein independently for each occurrence, XTEN is an extendedrecombinant polypeptide comprising greater than about 36 to about 3000amino acids with a substantially non-repetitive sequence wherein the sumof glycine (G), alanine (A), serine (S), threonine (T), glutamate (E)and proline (P) residues constitutes more than about 80% of the totalamino acid sequence of the XTEN, x is either 0 or 1, y is either 0 or 1,wherein x+y≥1, TM1 is a targeting moiety with specific binding affinityto a target selected from Table 1, TM2 is a targeting moiety withbinding affinity to a target selected from Table 1 or Table 2 that maybe identical or may be different to TM1, and L is a linker sequencehaving between 1 to about 300 amino acid residues. In one embodiment,the linker can be a sequence in which at least 80% of the residues arecomprised of amino acids glycine, serine, and/or glutamate, such as, butnot limited to a sequence with about 80-100% sequence identify to thesequence GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 1), or a portion ora multimer thereof.

In another embodiment, the invention provides an isolated binding fusionprotein of formula IIITM1-XTEN-TM2  IIIwherein independently for each occurrence XTEN is an extendedrecombinant polypeptide comprising greater than about 400 to about 3000amino acids with a substantially non-repetitive sequence wherein the sumof glycine (G), alanine (A), serine (S), threonine (T), glutamate (E)and proline (P) residues constitutes more than about 80% of the totalamino acid sequence of the XTEN, x is either 0 or 1, y is either 0 or 1,wherein x+y≥1, TM1 is a targeting moiety with specific binding affinityto a target ligand selected from Table 1 or Table 2; and TM2 is atargeting moiety with binding affinity to a target selected from Table 1or Table 2 that may be identical or may be different to the target boundby TM1.

In another embodiment, the invention provides a multivalent bindingfusion protein with three binding moieties of formula IV:(XTEN)_(x)-TM1-L1-TM2-L2-TM3-(XTEN)_(y)  IVwherein independently for each occurrence: XTEN is an extendedrecombinant polypeptide as described above, x is either 0 or 1; y iseither 0 or 1, wherein x+y≥1; TM1 is a targeting moiety with bindingaffinity to a target ligand selected from Table 1 or Table 2; TM2 is atargeting moiety with binding affinity to the target ligand selectedfrom Table 1 or Table 2 that may be identical or may be different toTM1; TM3 is a targeting moiety with binding affinity to the targetligand selected from Table 1 or Table 2 that may be identical or may bedifferent to either TM1 or TM2; L1 is a linker sequence having between 1to about 300 amino acid residues as described for formula II, andwherein the linker sequence is covalently bound to the C terminus of TM1and the N terminus of TM2; and L2 is a linker sequence that may beidentical to or different from L1, having between 1 to about 300 aminoacid residues as described as for formula II, and wherein the linkersequence is covalently bound to the C terminus of TM2 and the N terminusof TM3.

In some embodiments, the invention provides an isolated fusion proteinwith a single targeting moiety, wherein the targeting moiety exhibitsbinding specific affinity to a target selected from Table 1 or Table 2.In one embodiment of the foregoing, the target is selected from IL17,IL17R, RSV, HER2, IL12, IL23, RANKL, NGF, CD80, CD86, CD3, CD40, EGFR,TNFalpha, cMET, IL6R, and elastase. In other embodiments, the inventionprovides an isolated fusion protein with multiple targeting moieties(e.g., two, or three, or four, or five, or six, or seven or moretargeting moieties) wherein the targeting moiety exhibits bindingaffinity to one or more targets selected from Table 1 or Table 2. In oneembodiment of the foregoing, the one or more targets are selected fromIL17, IL17R, RSV, HER2, IL12, IL23, RANKL, NGF, CD80, CD86, CD3, CD40,EGFR, TNFalpha, cMET, IL6R, and elastase. In a preferred embodiment ofthe foregoing, the two or more targeting moieties are scFv. In any ofthe embodiments hereinabove described in this paragraph, the bindingaffinity constant (K_(d)) for the one or more targeting moieties of thesubject binding fusion protein and a target ligand is less than about10⁻⁵ M, alternatively less than about 10⁻⁶M, alternatively less thanabout 10⁻⁷M, alternatively less than about 10⁻⁸M, alternatively lessthan about 10⁻⁹M, or less than about 10⁻¹⁰ M, or less than about 10⁻¹¹M, or less than about 10⁻¹² M.

In some embodiments, binding fusion proteins exhibit an increasedapparent molecular weight as determined by size exclusionchromatography, compared to the actual molecular weight, wherein theapparent molecular weight is at least about 100 kD, 150 kD, 200 kD, 300kD, 400 kD, 500 kD, 600 kD, or 700 kD, while the actual molecular weightof the fusion protein is less than about 25 kD. Accordingly, the bindingfusion proteins can have an apparent molecular weight that is about4-fold greater, or about 5-fold greater, or about 6-fold greater, orabout 7-fold greater, or about 8-fold greater than the actual molecularweight of the binding fusion protein. In one embodiment, the isolatedbinding fusion protein of the foregoing embodiments exhibits an apparentmolecular weight factor under physiologic conditions that is greaterthan about 4, or about 5, or about 6, or about 7, or about 8.

In another embodiment, the invention provides isolated binding fusionprotein of any one of the preceding embodiments, further comprising oneor more molecules of a drug selected from Table 9. In one embodiment,the drug is covalently attached by a cross-linker to the XTEN,preferably through one or more cysteine or lysine amino acid residuesincorporated into the XTEN. In another embodiment, the drug iscovalently attached by a cross-linker to the targeting moiety,preferably through one or more cysteine or lysine amino acid residuesincorporated into the targeting moiety. The binding fusion protein drugconjugates can be in different configurations. In one embodiment, theinvention provides a binding fusion protein-drug conjugate compositionof formula V:[(D-CL)_(z1)-XTEN]_(x)-TM-[XTEN-(CL-D)_(z2)]_(y)  Vwherein independently for each occurrence: x is either 0 or 1; y iseither 0 or 1; XTEN is a cysteine- or lysine-engineered extendedrecombinant polypeptide as described above; TM is a targeting moietywith binding affinity to a target ligand selected from Table 1 or Table2 (which may comprise more than one binding domain joined by linkers);CL is a cross-linker as defined herein; D is a drug moiety selected fromTable 9 or a pharmaceutically acceptable salt, acid or derivativethereof; and z1 and z2 each are independently an integer from 1 to 100.Exemplary binding fusion protein-drug conjugate compositions of FormulaV can comprise XTEN that have from 1 to about 100 cysteine or lysineengineered amino acids, or from 1 to about 50 cysteine or lysineengineered amino acids, or from 1 to about 40 cysteine or lysineengineered amino acids, or from 1 to about 20 cysteine or lysineengineered amino acids, or from 1 to about 10 cysteine or lysineengineered amino acids, or from 1 to about 5 cysteine or lysineengineered amino acids that are available for conjugation to drugmolecules.

In another embodiment, the invention provides a binding fusionprotein-drug conjugate composition of formula VI:[(D-CL)_(z1)-XTEN]_(x)-TM1-L-TM2-[XTEN-(CL-D)_(z2)]_(y)  VIwherein independently for each occurrence: x is either 0 or 1, and y iseither 0 or 1; XTEN is a either a cysteine- or lysine-engineeredextended recombinant polypeptide as described; TM1 is a targeting moietywith binding affinity to a target ligand selected from Table 1 or Table2 (which may comprise more than one binding domain joined by linkers);TM2 is a targeting moiety with binding affinity to a target ligandselected from Table 1 or Table 2 (which may comprise more than onebinding domain joined by linkers) that may be identical or may bedifferent to TM1; and L is a linker sequence having between 1 to about300 amino acid residues wherein the linker sequence is covalently boundto the C terminus of TM1 and the N terminus of TM2; D is a drug moietyselected from Table 9 or a pharmaceutically acceptable salt, acid orderivative thereof; CL is a cross-linker as defined herein; and z1 andz2 each are independently an integer from 0 to 100. Exemplary bindingfusion protein-drug conjugate compositions of Formula VI can compriseXTEN that have from 1 to about 100 cysteine or lysine engineered aminoacids, or from 1 to about 50 cysteine or lysine engineered amino acids,or from 1 to about 40 cysteine or lysine engineered amino acids, or from1 to about 20 cysteine or lysine engineered amino acids, or from 1 toabout 10 cysteine or lysine engineered amino acids, or from 1 to about 5cysteine or lysine engineered amino acids that are available forconjugation to drug molecules.

The invention provides compositions of the isolated binding fusionproteins of any of the foregoing embodiments. In one embodiment, theinvention provides a pharmaceutical composition comprising a bindingfusion protein of any of the foregoing embodiments and at least onepharmaceutically acceptable carrier. In another embodiment, theinvention provides kits, comprising packaging material and at least afirst container comprising the pharmaceutical composition of theforegoing embodiment and a label identifying the pharmaceuticalcomposition and storage and handling conditions, and a sheet ofinstructions for the reconstitution and/or administration of thepharmaceutical compositions to a subject.

The invention further provides methods of use of the pharmaceuticalcompositions comprising the fusion protein of any of the foregoingembodiments in the treatment of a disease, disorder or condition in asubject in need thereof. In one embodiment of the method, the disease,disorder or condition is selected from the group consisting of breastcarcinoma, lung carcinoma, gastric carcinoma, esophageal carcinoma,colorectal carcinoma, liver carcinoma, ovarian carcinoma, thecoma,arrhenoblastoma, cervical carcinoma, endometrial carcinoma,endometriosis, fibrosarcoma, choriocarcinoma, head and neck cancer,nasopharyngeal carcinoma, laryngeal carcinoma, hepatoblastoma, Kaposi'ssarcoma, melanoma, skin carcinoma, hemangioma, cavernous hemangioma,hemangioblastoma, pancreas carcinoma, retinoblastoma, astrocytoma,glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma,neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas,urinary tract carcinoma, thyroid carcinoma, Wilm's tumor, renal cellcarcinoma, and prostate carcinoma.

In one embodiment, the invention provides a method of treating a diseaseor condition mediated by or associated with a target of Table 1 or Table2, comprising administering the pharmaceutical composition describedabove using a therapeutically effective amount to a subject in needthereof. In one embodiment, the target of Table 1 is a tumor associatedantigen, such as but not limited to the antigens of Table 2. In oneembodiment, the invention provides a method of treatment wherein thebinding fusion protein exhibits binding to the tumor associated antigenHER2. In a one embodiment of the method, the administration of thepharmaceutical composition using a therapeutically effective amount to asubject has a growth inhibitory effect on a tumor cell. The method oftreatment can comprise administering the pharmaceutical composition byan appropriate route, including subcutaneously, intramuscularly,intravitreally, or intravenously. In one embodiment, multipleconsecutive doses of the pharmaceutical composition are administered ata therapeutically effective dose regimen, and can result in animprovement in at least one measured parameter relevant for themetabolic disease, disorder or condition. In one embodiment, thetherapeutically effective dose regimen can be achieved using a twoadministrations of pharmaceutical composition per month dosing regimenfor the length of the dosing period. In one embodiment, thetherapeutically effective dose regimen is achieved using a oneadministration of pharmaceutical composition per month dosing regimenfor the length of the dosing period. In another embodiment, the bindingfusion protein has a growth inhibitory effect on SK-BR-3 cells in a cellculture assay.

The invention provides isolated nucleic acids comprising apolynucleotide sequence selected from (a) a polynucleotide encoding thebinding fusion protein of any of the foregoing embodiments, or (b) thecomplement of the polynucleotide of (a). In one embodiment of theforegoing, the isolated nucleic acid comprises a polynucleotide sequencethat has at least 80% sequence identity, or about 85%, or at least about90%, or about 91%, or about 92%, or about 93%, or about 94%, or about95%, or about 96%, or about 97%, or about 98%, or about 99% to about100% sequence identity to (a) a polynucleotide sequence that encodes apolypeptide selected from any one of Table 25, Table 40 or Table 41; or(b) the complement of the polynucleotide of (a). The invention providesexpression vectors comprising the nucleic acid of any of the embodimentshereinabove described in this paragraph. In one embodiment, theexpression vector of the foregoing further comprises a recombinantregulatory sequence operably linked to the polynucleotide sequence. Inanother embodiment, the polynucleotide sequence of the expressionvectors of the foregoing is fused in frame to a polynucleotide encodinga secretion signal sequence, which can be a prokaryotic signal sequence.In one embodiment, the secretion signal sequence is selected from OmpA,DsbA, and PhoA signal sequences.

The invention provides a host cell, which can comprise an expressionvector disclosed in the foregoing paragraph. In one embodiment, the hostcell is a prokaryotic cell, such as, but not limited to E. coli. Inanother embodiment, the host cell is a eukaryotic cell, such as, but notlimited to CHO.

The invention also provides host cells comprising an expression vector,wherein the expression vector encodes a binding fusion proteincomprising an N-terminal XTEN optimized for expression. In oneembodiment of the foregoing, the vector comprises a sequence thatencodes a polypeptide sequence that exhibits at least about 80%, morepreferably at least about 90%, more preferably at least about 91%, morepreferably at least about 92%, more preferably at least about 93%, morepreferably at least about 94%, more preferably at least about 95%, morepreferably at least about 96%, more preferably at least about 97%, morepreferably at least about 98%, more preferably at least 99%, or exhibits100% sequence identity to the amino acid sequence of AE48. AM48, AE624,AE912, or AM923. In one embodiment, the expression level of the encodedbinding fusion protein in the host cell is enhanced compared to theexpression level in a corresponding host cell comprising an expressionvector encoding a binding fusion protein lacking the N-terminal XTENoptimized for expression. In one embodiment, the expression level isenhanced at least about 50%, or about 75%, or about 100%, or about 150%,or about 200%, or about 400% compared to a corresponding binding fusionprotein not comprising the N-terminal XTEN sequence.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The features and advantages of the invention may be further explained byreference to the following detailed description and accompanyingdrawings that sets forth illustrative embodiments.

FIG. 1A-FIG. 1B show schematic representations of an exemplary scFvbinding fusion protein with a single targeting moiety depicted in an N-to C-terminus orientation. FIG. 1A shows the configuration of a scFvbinding fusion protein (100) with the single chain Fv targeting moietyin a “relaxed” conformation, comprising an N-terminal XTEN sequence(101), a VL binding domain sequence (102), a linker sequence (103), a VHbinding domain sequence (104), and an XTEN carrier sequence (105) at theC-terminus. FIG. 1B shows the flexible linker permitting the two bindingdomains to come into association to form the antigen binding site of thescFv targeting moiety.

FIG. 2A-FIG. 2B show schematic representations of exemplary componentsof a scFv binding fusion protein with two targeting moieties depicted inan N- to C-terminus orientation. FIG. 2A shows the configuration of ascFv binding fusion protein (107) with two single chain Fv targetingmoieties in a “relaxed” conformation, each comprising an N-terminal XTENsequence (101), a VL binding domain sequence (102), a linker sequence(103), a VH binding domain sequence (104), a second linker sequencejoining the first and the second targeting moieties (106) and an XTENcarrier sequence (105) at the C-terminus. FIG. 2B shows the flexiblelinkers permitting the two binding domains to come into association toform the antigen binding site of the two respective scFv targetingmoieties.

FIG. 3A-FIG. 3B show schematic representations of an exemplary monomericdiabody binding fusion protein that can form two antigen binding sites.FIG. 3A shows the configuration of a diabody binding fusion protein(108) with the two targeting moieties in a “relaxed” conformation, eachcomprising an N-terminal XTEN sequence (101), a first VL binding domainsequence (102), a short linker sequence joining the adjacent VL and VH(103), a first VH binding domain sequence (104), a second linkersequence joining the first and the second targeting moieties (106), asecond VH binding domain (109) intended to pair with the first VLbinding domain (104), a second VL (110) intended to pair with the firstVH (104) and an XTEN carrier sequence (105) at the C-terminus. FIG. 3Bshows the flexible middle linker permitting two binding domains (VL andVH) from the opposite ends of the molecule (rather than the two adjacentbinding domains that are constrained because of the short linker) tocome into association to form the two antigen binding sites, resultingin two diabody targeting moieties within the monomeric binding fusionprotein.

FIG. 4A-FIG. 4B show schematic representations of an exemplary VEGFcytokine binding fusion protein. FIG. 4A shows the configuration of thebinding fusion protein (114) with the dimer binding regions in a“relaxed” conformation, each comprising an N-terminal XTEN sequence(101), a first D2 domain sequence (111), a short linker sequence joiningthe adjacent domain units (103), a first D3 domain sequence (112), asecond flexible linker sequence joining the first and the second bindingdomains (106), a second VH binding domain (109) intended to pair withthe first VL binding domain (104), a second D2 domain (111), a shortlinker sequence joining the adjacent domain units (103), a second D3domain sequence (112), and an XTEN carrier sequence (105) at theC-terminus. In the schematic, the dimeric VEGF target (113) has begun toassociate with one of the binding domains.

FIG. 4B shows the flexible middle linker permitting two binding domainsof the molecule to surround the dimeric VEGF (113) to form the bindingcomplex that sequesters the VEGF molecule.

FIG. 5 shows schematic representations of an exemplary receptor bindingfusion protein comprising Ig-like binding domains. The binding fusionprotein (115) has an N-terminal XTEN sequence (101) and in this case,two Ig-like binding regions (116), a flexible linker sequence joiningthe adjacent Ig-like binding regions (106) that would permit the twobinding regions to sequester the target ligand, and an XTEN carriersequence (105) at the C-terminus.

FIG. 6A-FIG. 6D show schematic representations of exemplary genes thatencode binding fusion proteins, all depicted in a 5′ to 3′ orientation,with all component sequences linked in frame. FIG. 6A shows theconfiguration of a gene encoding scFv binding fusion proteins (100) intwo configurations (5′ to 3′) with a single targeting moiety, with thefirst having nucleotides encoding a VL binding domain sequence (202), alinker sequence (203), a VH binding domain sequence (204), and an XTENcarrier sequence (205) at the 3′-end. Below that is the oppositeconfiguration, with nucleotides encoding an N-terminal XTEN (201),linked to nucleotides encoding a VL binding domain sequence (202), alinker sequence (203), and a VH binding domain sequence FIG. 6B showsthe configuration of a gene encoding a diabody binding fusion proteinthat can form two antigen binding sites (208), with the top constructshown comprising, at the 5′ end, genes for a first VL binding domainsequence (202), a short linker sequence joining the adjacent VL and VHgenes (303), a first VH binding domain sequence (204), a second linkersequence (that can be an XTEN) joining the first and the secondtargeting moieties (206), a second VH binding domain (209), a second VLbinding domain (210) and an XTEN carrier sequence (205) at the 3′ end.The lower gene encodes, at the 5′ end, an N-terminal XTEN (201), a firstVL binding domain sequence (202), a short linker sequence joining theadjacent VL and VH genes (203), a first VH binding domain sequence(204), a second linker sequence (than can be an XTEN) joining the firstand the second targeting moieties (206), a second VH binding domain(209), a second VL binding domain (210) and an XTEN carrier sequence(205) at the 3′ end. FIG. 6C shows the configuration of a gene encodinga cytokine binding protein. The gene encodes, at the 5′ end, anN-terminal XTEN (201), a first D2 binding domain sequence (211), a shortlinker sequence joining the adjacent domain genes (203), a first D3binding domain sequence (212), a second linker sequence (206) (than canbe an XTEN) joining the first and the second targeting moieties, asecond D2 binding domain (211), a second D3 binding domain (212) and anXTEN carrier sequence (205) at the 3′ end. FIG. 6D shows threeconfigurations, 5′ to 3′, of genes encoding a domain antibody bindingfusion protein (217). The genes encode, where shown, an N-terminal XTEN(201), the VHH binding domain sequence (218), and an XTEN carriersequence (205) at the 3′ end.

FIG. 7 is a schematic flowchart of representative steps in the assembly,production and the evaluation of a XTEN.

FIG. 8 is a schematic flowchart of representative steps in the assemblyof a targeting moiety-XTEN polynucleotide construct encoding, in thiscase, an anti-Her2 binding fusion protein. Individual oligonucleotides501 are annealed into sequence motifs 502 such as a 12 amino acid motif(“12-mer”), which is ligated to additional sequence motifs from alibrary to create a pool that encompasses the desired length of the XTEN504, as well as ligated to a smaller concentration of an oligocontaining BbsI, and KpnI restriction sites 503. The resulting pool ofligation products is gel-purified and the band with the desired lengthof XTEN is cut, resulting in an isolated XTEN gene with a stoppersequence 505. The XTEN gene is cloned into a stuffer vector. In thiscase, the vector encodes an optional CBD sequence 506 and a GFP gene508. Digestion is than performed with BbsI/HindIII to remove 507 and 508and place the stop codon. The resulting product is then cloned into aBsaI/HindIII digested vector containing a gene encoding the svFvanti-Her2, resulting in the gene 500 encoding an binding fusion protein.

FIG. 9 is a schematic flowchart of representative steps in the assemblyof a gene encoding a binding fusion protein comprising a targetingmoiety and XTEN, its expression and recovery as a fusion protein, andits evaluation as a candidate binding fusion protein product.

FIG. 10A-FIG. 10D is a schematic representation of the design ofanti-Her2 binding fusion protein expression vectors with differentprocessing strategies. FIG. 10A shows an expression vector encoding XTENfused to the 3′ end of the sequence encoding anti-Her2 binding moiety.Note that no additional leader sequences are required in this vector.FIG. 10B depicts an expression vector encoding XTEN fused to the 5′ endof the sequence encoding anti-Her2 binding moiety with a CBD leadersequence and a TEV protease site. FIG. 10C depicts an expression vectoras in FIG. 10B where the CBD and TEV processing site have been replacedwith an optimized N-terminal leader sequence (NTS). FIG. 10D depicts anexpression vector encoding an NTS sequence, an XTEN, a sequence encodinganti-Her2 binding moiety, and then a second sequence encoding an XTEN.

FIG. 11 shows results of expression assays for the indicated constructscomprising GFP and XTEN sequences, conducted as described in Example 14.The expression cultures were assayed using a fluorescence plate reader(excitation 395 nm, emission 510 nm) to determine the amount of GFPreporter present and the results are graphed as box and whisker plots.

FIG. 12 shows three randomized libraries used for the third and fourthcodons in the N-terminal sequences of clones from LCW546, LCW547 andLCW552. The libraries were designed with the third and fourth residuesmodified such that all combinations of allowable XTEN codons werepresent at these positions, as shown. In order to include all theallowable XTEN codons for each library, nine pairs of oligonucleotidesencoding 12 amino acid motifs with codon diversities of third and fourthresidues were designed, annealed and ligated into the NdeI/BsaIrestriction enzyme digested stuffer vector pCW0551(Stuffer-XTEN_AM875-GFP), and transformed into E. coli BL21Gold(DE3)competent cells to obtain colonies of the three libraries LCW0569 (SEQID NOS 863-864), LCW0570 (SEQ ID NOS 865-866), and LCW0571 (SEQ ID NOS867-868).

FIG. 13 shows a histogram of a retest of the top 75 clones after theoptimization step, as described in Example 15, for GFP fluorescencesignal relative to the benchmark CBD_AM875 construct. The resultsindicated that several clones were now superior to the benchmark clonesseen in FIG. 11.

FIG. 14 is a schematic of a combinatorial approach undertaken for theunion of codon optimization preferences for two regions of theN-terminus 48 amino acids. The approach created novel 48mers at theN-terminus of the XTEN protein for evaluation of the optimization ofexpression for leader sequences to enhance expression of XTEN proteinswhere the XTEN is N-terminal to the targeting moieties.

FIG. 15 shows an SDS-PAGE gel confirming expression of preferred clonesobtained from the XTEN N-terminal codon optimization experiments, incomparison to benchmark XTEN clones comprising CBD leader sequences atthe N-terminus of the construct sequences.

FIG. 16A-FIG. 16C show an SDS-PAGE gel of samples from a stability studyof the fusion protein of XTEN_AE864 fused to the N-terminus of GFP. FIG.16A shows the results after in vitro incubation of GFP-XTEN incynomolgus monkey plasma and FIG. 16C shows the results after incubationin rat kidney lysate for up to 7 days at 37° C. with samples analyzed bySDS PAGE followed by detection using Western analysis and detection withantibodies against GFP, as described in Example 56. In addition, FIG.16B shows the results of GFP-XTEN administered to cynomolgus monkeys inwhich samples were withdrawn at 0, 1 and 7 days and analyzed by SDS PAGEfollowed by detection using Western analysis and detection withantibodies against GFP.

FIG. 17A-FIG. 17C show the characterization of multivalent scFv bindingfusion proteins. FIG. 17A is a schematic representation of twovariations of multivalent scFv binding fusion proteins, with amonospecific construct on the left comprising two targeting moietiesdirected to HER2 and a bispecific construct on the right comprising atargeting moiety to HER2 and a second targeting moiety to EGFR. FIG. 17Bshows an SDS-PAGE of materials following purification, as described inExample 29. Lane 1 shows the molecular weight standards, lane 2 showsthe purified aHER2-XTEN-aHER2, and lane 3 shows the purified bispecificaHER2-XTEN-aEGFR. FIG. 17C shows the results of SEC analysis ofaHER2-XTEN-aEGFR compared to molecular weight standards, anddemonstrates that no dimers or other higher-order oligomers are formedand that the protein has an approximate apparent molecular weight ofapproximately 500 kDa, as described in Example 37.

FIG. 18 shows results of a binding activity assay of aHER2-XTEN-aEGFRbispecific binding fusion protein to its respective targets using anELISA format, as described in Example 37.

FIG. 19A-FIG. 19B show a schematic of two scFv binding fusion proteinconstructs with a GFP tag and flow cytometry results of cell bindingassays. FIG. 19A is a schematic of aCD3-XTEN-GFP and aHER2-XTEN-GFPconstructs. FIG. 19B shows the output of flow-cytometry in which the twoconstructs were individually reacted with Jurkat CD3+ and SK-BR3 HER2+cells, as described in Example 36, demonstrating the binding specificityof the respective constructs towards their ligands and the lack ofbinding to the heterologous targets.

FIG. 20A-FIG. 20B show results from characterization assays of abispecific scFv binding fusion protein, as described in Example 36. FIG.20A shows an SDS-PAGE gel of the purified aHER2-aCD3-XTEN. FIG. 20Bshows the output of a size exclusion chromatography (SEC) analysis ofthe aHER2-aCD3-XTEN compared to molecular weight standards, anddemonstrates that no dimers or other higher-order oligomers are formedand that the protein has an approximate apparent molecular weight ofapproximately 500 kDa, approximately five-fold higher than the massindicated in the SDS-PAGE assay of FIG. 20A.

FIG. 21A-FIG. 21C show results from characterization assays of abispecific scFv binding fusion protein, as described in Example 36. FIG.21A shows results of a binding assay performed by ELISA comparing a scFvof anti-Her2 on the N-terminus of a binding fusion protein to abispecific scFv of anti-CD3 on the N-terminus and anti-Her2 on theC-terminus of an XTEN, against wells coated with HER2, showing that thebispecific retains binding affinity to the HER2 target. FIGS. 21B and Cshow results of a flow cytometry cell binding assays using the mono- andbispecific constructs, respectively, against HER2-expressing SK-BR-3cells. The results show greater signal for the N-terminal anti-HersscFv-XTEN (FIG. 21B) than the bispecific construct, consistent with thatof the ELISA study, but that the bispecific construct neverthelessretains good binding activity.

FIG. 22 shows results from a flow cytometry assay to characterize thebinding of aCD3-XTEN-GFP to CD3-positive Jurkat cells. The assay wasperformed using anti-GFP antibody detection of aCD3-XTEN-GFP reacted inthe presence of a 10-fold molar excess of aHER2-aCD3-XTEN, as describedin Example 36. The results show that excess bispecific aHER2-aCD3-XTENcompetitively displaces the monospecific aCD3-XTEN-GFP protein andeliminates the observed MFI shift.

FIG. 23 shows results from a tumor cell killing assay in which varyingconcentrations of the bispecific aHER2-XTEN-aCD3 were incubated witheither M21 or SK-BR-3 target cells for 24 h, as described in Example 36,followed by staining with propidium iodide to measure killing, shown inthe bar graph as the proportion of dead tumor cells for the two celllines.

FIG. 24A-FIG. 24B show schematic representations of single andmultivalent Vhh binding fusion protein constructs and theircharacterization. FIG. 24A shows schematic portrayals of monomeric,dimeric, tetrameric, and hexameric anti-EGFR Vhh constructs linked byXTEN, with a C-terminal GFP. FIG. 24B show SDS-PAGE of lysate and thepurified Vhh constructs, as described in Example 38, demonstrating thepurity and the “ladder” increase in molecular weight with increasingunits of the anti-EGFR Vhh targeting moiety.

FIG. 25 show the output of a size exclusion chromatography (SEC)analysis of the monomeric and multivalent anti-EGFR Vhh binding fusionprotein constructs of FIG. 24A compared to molecular weight standards,as described in Example 38. The results demonstrate proportionalincreases approximate apparent molecular weight of the constructs withincreasing numbers of Vhh targeting moieties, compared to the molecularweights determined by SDS-PAGE in FIG. 24B and listed in Table 26.

FIG. 26A-FIG. 26B show results of binding characterization ELISA assaysof monomeric and multivalent targeting moiety anti-EGFR Vhh bindingfusion protein constructs, as described in Example 38 (the constructsdepicted schematically in FIG. 24A). FIG. 26A shows results of the ELISAsignal generated using equi-molar concentrations of the variousmonomeric or multivalent anti-EGFR Vhh binding fusion proteins againstthe EGFR target. FIG. 26B shows the results of binding curves for thesame constructs at various dilutions, with the multivalent formsresulting in more signal than the construct with a single Vhh targetingmoiety.

FIG. 27 shows the results of a binding characterization ELISA assay oftwo anti-CD40 scFv binding fusion protein constructs, AC384 (closedsquares) and AC385 (open squares), against human CD40, as described inExample 34.

FIG. 28A-FIG. 28B show the results of characterization assays for ananti-IL6R binding fusion protein. FIG. 28A shows the uniformity of thepurified protein assessed by SEC, which showed a monodispersed peak withminimal contamination. FIG. 28B shows results of an ELISA binding assayof the anti-IL6R binding fusion protein against human IL6R, as describedin Example 33.

FIG. 29 shows the results of a binding characterization ELISA assay ofanti-Her2 binding protein with two anti-Her2 targeting moieties againsthuman HER2, as described in Example 35.

FIG. 30 shows the near UV circular dichroism spectrum of Ex4-XTEN_AE864,performed as described in Example 52.

FIG. 31 shows the pharmacokinetic profile (plasma concentrations) incynomolgus monkeys after single doses of different compositions of GFPlinked to unstructured polypeptides of varying length, administeredeither subcutaneously or intravenously, as described in Example 53. Thecompositions were GFP-L288, GFP-L576, GFP-XTEN_AF576, GFP-Y576 andXTEN_AD836-GFP. Blood samples were analyzed at various times afterinjection and the concentration of GFP in plasma was measured by ELISAusing a polyclonal antibody against GFP for capture and a biotinylatedpreparation of the same polyclonal antibody for detection. Results arepresented as the plasma concentration versus time (h) after dosing andshow, in particular, a considerable increase in half-life for theXTEN_AD836-GFP, the composition with the longest sequence length ofXTEN. The construct with the shortest sequence length, the GFP-L288 hadthe shortest half-life.

FIG. 32 shows results of a size exclusion chromatography analysis ofglucagon-XTEN construct samples measured against protein standards ofknown molecular weight, with the graph output as absorbance versusretention volume, as described in Example 60. The glucagon-XTENconstructs are 1) glucagon-Y288; 2) glucagonY-144; 3) glucagon-Y72; and4) glucagon-Y36. The results indicate an increase in apparent molecularweight with increasing length of the XTEN component.

FIG. 33 shows overlays of SEC chromatograms of threeaHer2-XTEN(Cys)-AF680 conjugated fusion proteins with three differentXTEN; AE864, AE576, and AE288 (top to bottom), performed as described inExample 41. The chromatograms show that the Alexa Fluor 680 wassuccessfully conjugated to the aHer2-XTEN(Cys) protein as the retentiontime of the absorbance of the Alexa Fluor 680 (A690) overlaps with thatof the aHer2-XTEN(Cys) protein (A280) for all three samples, and thatthe constructs eluted in a proportional fashion relative to length ofthe XTEN component. Note that dye retention time is significantlyshifted from the expected elution time of free dye at ˜52 minutes.Additionally, the relative lack of material that elutes ahead of thevarious aHer2-XTEN(Cys) peaks indicates a lack of aggregation and amonodispersed product.

FIG. 34A-FIG. 34C show output of flow cytometry assays for the threefusion proteins described in FIG. 33 compared to unlabeled Herceptin andcontrol IgG-AF680 conjugate, measuring forward and side scatter vs. FL4for Alexa680, as described in Example 41. FIG. 34A is the output of theaHer2-XTEN_AE864-AF680 assay. FIG. 34B is the output of theaHer2-XTEN_AE576-AF680 assay, and FIG. 34C is the output of theaHer2-XTEN_AE288-AF680 assay.

FIG. 35 shows the graphed results of in vivo imaging data from femalenu/nu mice bearing SKOV3 tumor cells given a single injection of high orlow dose aHer2-XTEN-AE-288-Cys-AF680, aHer2-XTEN-AE-576-Cys-AF680,aHer2-XTEN-AE-864-Cys-AF680 or Herceptin-AF680 control, as described inExample 41.

FIG. 36 shows the graphed results of ex vivo imaging data from the sametreatment groups as per FIG. 36, demonstrating that all aHer2-XTENbinding fusion protein constructs had penetration into the assayedtissues, with the longer AE864 XTEN construct demonstrating the highestlevels compared to the other two, as described in Example 41.

FIG. 37 is a schematic of the logic flow chart of the algorithmSegScore. In the figure the following legend applies: i, j—counters usedin the control loops that run through the entire sequence; HitCount—thisvariable is a counter that keeps track of how many times a subsequenceencounters an identical subsequence in a block; SubSeqX—this variableholds the subsequence that is being checked for redundancy; SubSeqY—thisvariable holds the subsequence that the SubSeqX is checked against;BlockLen—this variable holds the user determined length of the block;SegLen—this variable holds the length of a segment. The program ishardcoded to generate scores for subsequences of lengths 3, 4, 5, 6, 7,8, 9, and 10; Block—this variable holds a string of length BlockLen. Thestring is composed of letters from an input XTEN sequence and isdetermined by the position of the i counter; SubSeqList—this is a listthat holds all of the generated subsequence scores.

FIG. 38 depicts the application of the algorithm SegScore to ahypothetical XTEN of 11 amino acids in order to determine therepetitiveness. An XTEN sequence (SEQ ID NO: 869) consisting of N aminoacids is divided into N-S+1 subsequences of length S (S=3 in this case).A pair-wise comparison of all subsequences is performed and the averagenumber of identical subsequences is calculated to result, in this case,in a subsequence score of 1.89.

FIG. 39A-FIG. 39E illustrate the use of donor XTEN sequences to producetruncated XTEN sequences. FIG. 39A provides the sequence of AG864 (SEQID NO: 870), with the underlined sequence used to generate an AG576 (SEQID NO: 871) sequence. FIG. 39B provides the sequence of AG864 (SEQ IDNO: 872), with the underlined sequence used to generate an AG288 (SEQ IDNO: 873) sequence. FIG. 39C provides the sequence of AG864 (SEQ ID NO:874), with the underlined sequence used to generate an AG144 (SEQ ID NO:875) sequence. FIG. 39D provides the sequence of AE864 (SEQ ID NO: 876),with the underlined sequence used to generate an AE576 (SEQ ID NO: 877)sequence. FIG. 39E provides the sequence of AE864 (SEQ ID NO: 878), withthe underlined sequence used to generate an AE288 (SEQ ID NO: 879)sequence.

FIG. 40A-FIG. 40D show various schematic examples of XTEN-basedprotein-drug conjugates, with the chemically conjugated drug-crosslinkerligand designated “D”. FIG. 40A shows three XTEN-drug conjugates, with1, 2 and 4 drug molecules conjugated to the XTEN. FIG. 40B shows fourconfigurations of BFP-D with the binding domain (“BD”) linked to the N-or C-terminus of XTEN of the fusion protein and either 1, 3 or 4 drugmolecules conjugated to the XTEN carrier by cross-linkers. FIG. 40Cshows four configurations of BFP-D with the scFv binding domain on theN- or C-terminus of the XTEN and either 1, 3 or 4 drug moleculesconjugated to the XTEN carrier by cross-linkers. FIG. 40D shows fourconfigurations of multivalent BFP-D with two binding domains (“BD”) onthe N- or C-terminus or configurations with an N-terminal XTEN andeither 1, 2, 3 or 4 drug molecules conjugated to the XTEN carrier orN-terminal XTEN by cross-linkers.

FIG. 41A-FIG. 41D show various schematic examples of the conjugationprocess to make conjugates with multiple drug ligands using orthogonalcoupling chemistries. FIG. 41A shows a two-step process of coupling ofone drug-crosslinker ligand (D1) to an internal cysteine of acysteine-engineered XTEN and a second, different drug-crosslinker ligand(D2) to the N-terminus of XTEN.

FIG. 41B shows a two-step process of coupling of one ligand to aninternal cysteine cysteine-engineered XTEN and two ligands to aminogroups in a recombinant binding domain (BD). FIG. 41C shows a two-stepprocess of coupling of two drug ligands (D1) to two internal cysteine ofa cysteine-engineered XTEN and a second drug ligand (D2) to theN-terminus of XTEN. FIG. 41D shows a two-step process of coupling twodrug ligands (D1) to XTEN internal cysteines and 1 drug-crosslinkerligand (D2) to the N-terminus and two additional D2 drug-crosslinkerligands to internal lysine residues of the engineered XTEN.

FIG. 42A-FIG. 42B show results of analytical assays of XTEN conjugatedwith cross-linked FITC, as described in Example 63. FIG. 42A shows theco-migration in a gel imaged by UV light box to show the large apparentMW of FITC-containing conjugated species, also detected by SEC at OD214(protein signal) and OD495 (FITC signal) in a SEC column, indicatingsuccessful labeling of the XTEN with minimal free dye contamination. Thematerials by lane (left to right, after the MW standards are: labeledFITC-CL-CBD-XTEN; labeled FITC-CL-XTEN; purified FITC-CL-XTEN; purifiedFITC-CL-XTEN; and purified FITC-CL-XTEN. The gel was imaged by UV lightbox to show FITC apparent MW of FITC containing species. FIG. 42B showsthe results of SEC analysis of FITC-conjugated XTEN, showing the overlapof the output of materials detected at OD214 and OD495, and also theapparent large molecular weight.

FIG. 43 shows results of SEC analyses of the peak elution fractions ofconjugates of GFP cross-linked to XTEN and free GFP, as described inExample 64. Cross-linking was confirmed by co-migration of the OD214protein signal and OD395 GFP signal in the SEC column.

FIG. 44 shows the results of pharmacokinetic assays of GFP-X-XTEN andFITC-X-XTEN tested in cynomolgus monkeys, as described in Example 65.

FIG. 45 shows a schematic of the orthogonal chemistry process to createa BFP-D comprising a scFv anti-HER2 with paclitaxel conjugated to theXTEN, resulting in aHER2-XTEN-CL-paclitaxel. The paclitaxel is firstreacted with an activated linker, then conjugated to acysteine-engineered XTEN, as described in Example 66.

DETAILED DESCRIPTION OF THE INVENTION

Before the embodiments of the invention are described, it is to beunderstood that such embodiments are provided by way of example only,and that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Numerous variations, changes, and substitutions will nowoccur to those skilled in the art without departing from the invention.

Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

The terms “polypeptide”, “peptide”, and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified, forexample, by disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component.

As used herein the term “amino acid” refers to either natural and/orunnatural or synthetic amino acids, including but not limited to glycineand both the D or L optical isomers, and amino acid analogs andpeptidomimetics. Standard single or three letter codes are used todesignate amino acids.

The term “natural L-amino acid” means the L optical isomer forms ofglycine (G), proline (P), alanine (A), valine (V), leucine (L),isoleucine (I), methionine (M), cysteine (C), phenylalanine (F),tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R),glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D),serine (S), and threonine (T).

The term “non-naturally occurring,” as applied to sequences and as usedherein, means polypeptide or polynucleotide sequences that do not have acounterpart to, are not complementary to, or do not have a high degreeof homology with a wild-type or naturally-occurring sequence found in amammal. For example, a non-naturally occurring polypeptide or fragmentmay share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or evenless amino acid sequence identity as compared to a natural sequence whensuitably aligned.

The terms “hydrophilic” and “hydrophobic” refer to the degree ofaffinity that a substance has with water. A hydrophilic substance has astrong affinity for water, tending to dissolve in, mix with, or bewetted by water, while a hydrophobic substance substantially lacksaffinity for water, tending to repel and not absorb water and tendingnot to dissolve in or mix with or be wetted by water Amino acids can becharacterized based on their hydrophobicity. A number of scales havebeen developed. An example is a scale developed by Levitt, M, et al., JMol Biol (1976) 104:59, which is listed in Hopp, T P, et al., Proc NatlAcad Sci USA (1981) 78:3824. Examples of “hydrophilic amino acids” arearginine, lysine, threonine, alanine, asparagine, and glutamine. Ofparticular interest are the hydrophilic amino acids aspartate,glutamate, and serine, and glycine. Examples of “hydrophobic aminoacids” are tryptophan, tyrosine, phenylalanine, methionine, leucine,isoleucine, and valine.

A “fragment” is a truncated form of a native biologically active proteinthat retains at least a portion of the therapeutic and/or biologicalactivity. A “variant” is a protein with sequence homology to the nativebiologically active protein that retains at least a portion of thetherapeutic and/or biological activity of the biologically activeprotein. For example, a variant protein may share at least 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identitywith the reference biologically active protein. As used herein, the term“biologically active protein moiety” includes proteins modifieddeliberately, as for example, by site directed mutagenesis, insertions,or accidentally through mutations.

A “host cell” includes an individual cell or cell culture which can beor has been a recipient for the subject vectors. Host cells includeprogeny of a single host cell. The progeny may not necessarily becompletely identical (in morphology or in genomic of total DNAcomplement) to the original parent cell due to natural, accidental, ordeliberate mutation. A host cell includes cells transfected in vivo witha vector of this invention.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. As is apparent to those of skill in the art,a non-naturally occurring polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, does not require “isolation” todistinguish it from its naturally occurring counterpart. In addition, a“concentrated”, “separated” or “diluted” polynucleotide, peptide,polypeptide, protein, antibody, or fragments thereof, is distinguishablefrom its naturally occurring counterpart in that the concentration ornumber of molecules per volume is generally greater than that of itsnaturally occurring counterpart. In general, a polypeptide made byrecombinant means and expressed in a host cell is considered to be“isolated.”

An “isolated” polynucleotide or polypeptide-encoding nucleic acid orother polypeptide-encoding nucleic acid is a nucleic acid molecule thatis identified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal or extra-chromosomal location differentfrom that of natural cells.

A “chimeric” protein contains at least one fusion polypeptide comprisingregions in a different position in the sequence than that which occursin nature. The regions may normally exist in separate proteins and arebrought together in the fusion polypeptide; or they may normally existin the same protein but are placed in a new arrangement in the fusionpolypeptide. A chimeric protein may be created, for example, by chemicalsynthesis, or by creating and translating a polynucleotide in which thepeptide regions are encoded in the desired relationship.

“Conjugated” and “conjugation” refers to the covalent joining togetherof two or more chemical elements or components by chemical reaction,rather than recombinantly; e.g., a drug and an XTEN.

A “cross-linker” or “CL” means a chemical moiety comprising a covalentbond, drug-linker, or a chain of atoms that covalently conjugate a drugmoiety to a protein. Cross-linker components can comprise one or tworeactive groups to facilitate the conjugation of a drug and a protein,and the reactive groups can have been blocked by protecting groups topermit the selective conjugation with a given drug or protein reactant.

A “reactive group” is a chemical structure or functional group that ispart of or that can be coupled to a reactant for conjugation. Examplesfor reactive groups are amino groups, carboxyl groups, sulfhydrylgroups, hydroxyl groups, aldehyde groups, azide groups. Some reactivegroups can be activated to facilitate coupling with a second reactivegroup, such as a cross-linker component. Examples for activation are thereaction of a carboxyl group with carbodiimide, the conversion of acarboxyl group into an activated ester, the conversion of a carboxylgroup into an azide function, or the conversion of a hydroxyl to athiol.

The term “cytotoxic agent” as used herein refers to a substance thatcauses destruction of cells. The term is intended to include radioactiveisotopes, drugs, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including synthetic analogs and derivatives thereof.

The term “cytostatic agent” refers to a substance that has the effect oflimiting the function of cells, such as limiting cellular growth orproliferation of cells.

A “drug” is a non-protein chemical compound useful in the treatment of adisease, disorder or condition in a subject; e.g., cancer, inflammatorydiseases or conditions such as rheumatoid arthritis, metabolic disorderssuch as diabetes, infectious diseases, diseases of the organs such asasthma, or generalized conditions such as pain and hypertension, etc.Administration of a drug to a subject or to a cell can, for example,result in a pharmacologic effect, a therapeutic effect, an inhibitoryeffect, or result in cell death.

In the context of polypeptides, “fusion”, “fused” and “linked,” are usedinterchangeably herein. These terms refer to the joining together of twomore protein components, by whatever means including chemicalconjugation or recombinant means. For example, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe sequence. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous, and in reading phase or in-frame. An“in-frame fusion” refers to the joining of two or more open readingframes (ORFs) to form a continuous longer ORF, in a manner thatmaintains the correct reading frame of the original ORFs. Thus, theresulting recombinant fusion protein is a single protein containing twoore more segments that correspond to polypeptides encoded by theoriginal ORFs (which segments are not normally so joined in nature).

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminus direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide. A“partial sequence” is a linear sequence of part of a polypeptide that isknown to comprise additional residues in one or both directions.

“Heterologous” means derived from a genotypically distinct entity fromthe rest of the entity to which it is being compared. For example, aglycine rich sequence removed from its native coding sequence andoperatively linked to a coding sequence other than the native sequenceis a heterologous glycine rich sequence. The term “heterologous” asapplied to a polynucleotide, a polypeptide, means that thepolynucleotide or polypeptide is derived from a genotypically distinctentity from that of the rest of the entity to which it is beingcompared.

The terms “polynucleotides”, “nucleic acids”, “nucleotides” and“oligonucleotides” are used interchangeably. They refer to a polymericform of nucleotides of any length, either deoxyribonucleotides orribonucleotides, or analogs thereof. Polynucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The following are non-limiting examples of polynucleotides:coding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.

The term “complement of a polynucleotide” denotes a polynucleotidemolecule having a complementary base sequence and reverse orientation ascompared to a reference sequence, such that it could hybridize with areference sequence with complete fidelity.

“Recombinant” as applied to a polynucleotide means that thepolynucleotide is the product of various combinations of in vitrocloning, restriction and/or ligation steps, and other procedures thatresult in a construct that can potentially be expressed in a host cell.

The terms “gene” or “gene fragment” are used interchangeably herein.They refer to a polynucleotide containing at least one open readingframe that is capable of encoding a particular protein after beingtranscribed and translated. A gene or gene fragment may be genomic orcDNA, as long as the polynucleotide contains at least one open readingframe, which may cover the entire coding region or a segment thereof. A“fusion gene” is a gene composed of at least two heterologouspolynucleotides that are linked together.

“Homology” or “homologous” refers to sequence similarity orinterchangeability between two or more polynucleotide sequences or twoor more polypeptide sequences. When using a program such as BestFit todetermine sequence identity, similarity or homology between twodifferent amino acid sequences, the default settings may be used, or anappropriate scoring matrix, such as blosum45 or blosum80, may beselected to optimize identity, similarity or homology scores.Preferably, polynucleotides that are homologous are those whichhybridize under stringent conditions as defined herein and have at least70%, preferably at least 80%, more preferably at least 90%, morepreferably 95%, more preferably 97%, more preferably 98%, and even morepreferably 99% sequence identity to those sequences.

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a polynucleotide willhybridize to its target sequence, to a detectably greater degree thanother sequences (e.g., at least 2-fold over background). Generally,stringency of hybridization is expressed, in part, with reference to thetemperature and salt concentration under which the wash step is carriedout. Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short polynucleotides (e.g., 10to 50 nucleotides) and at least about 60° C. for long polynucleotides(e.g., greater than 50 nucleotides)—for example, “stringent conditions”can include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C.,and three washes for 15 min each in 0.1×SSC/1% SDS at 60° C. to 65° C.Alternatively, temperatures of about 65° C., 60° C., 55° C., or 42° C.may be used. SSC concentration may be varied from about 0.1 to 2×SSC,with SDS being present at about 0.1%. Such wash temperatures aretypically selected to be about 5° C. to 20° C. lower than the thermalmelting point © for the specific sequence at a defined ionic strengthand pH. The Tm is the temperature (under defined ionic strength and pH)at which 50% of the target sequence hybridizes to a perfectly matchedprobe. An equation for calculating Tm and conditions for nucleic acidhybridization are well known and can be found in Sambrook, J. et al.(1989) Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3,Cold Spring Harbor Press, Plainview N.Y.; specifically see volume 2 andchapter 9. Typically, blocking reagents are used to block non-specifichybridization. Such blocking reagents include, for instance, sheared anddenatured salmon sperm DNA at about 100-200 μg/ml. Organic solvent, suchas formamide at a concentration of about 35-50% v/v, may also be usedunder particular circumstances, such as for RNA:DNA hybridizations.Useful variations on these wash conditions will be readily apparent tothose of ordinary skill in the art.

The terms “percent identity” and “% identity,” as applied topolynucleotide sequences, refer to the percentage of residue matchesbetween at least two polynucleotide sequences aligned using astandardized algorithm. Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences. Percent identity may bemeasured over the length of an entire defined polynucleotide sequence,for example, as defined by a particular SEQ ID number, or may bemeasured over a shorter length, for example, over the length of afragment taken from a larger, defined polynucleotide sequence, forinstance, a fragment of at least 45, at least 60, at least 90, at least120, at least 150, at least 210 or at least 450 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

“Percent (%) amino acid sequence identity,” with respect to thepolypeptide sequences identified herein, is defined as the percentage ofamino acid residues in a query sequence that are identical with theamino acid residues of a second, reference polypeptide sequence or aportion thereof, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared. Percentidentity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defined polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 70 or at least 150 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

The term “non-repetitiveness” as used herein in the context of apolypeptide refers to a lack or limited degree of internal homology in apeptide or polypeptide sequence. The term “substantially non-repetitive”can mean, for example, that there are few or no instances of fourcontiguous amino acids in the sequence that are identical amino acidtypes or that the polypeptide has a subsequence score (defined infra) of10 or less or that there isn't a pattern in the order, from N- toC-terminus, of the sequence motifs that constitute the polypeptidesequence. The term “repetitiveness” as used herein in the context of apolypeptide refers to the degree of internal homology in a peptide orpolypeptide sequence. In contrast, a “repetitive” sequence may containmultiple identical copies of short amino acid sequences. For instance, apolypeptide sequence of interest may be divided into n-mer sequences andthe number of identical sequences can be counted. Highly repetitivesequences contain a large fraction of identical sequences whilenon-repetitive sequences contain few identical sequences. In the contextof a polypeptide, a sequence can contain multiple copies of shortersequences of defined or variable length, or motifs, in which the motifsthemselves have non-repetitive sequences, rendering the full-lengthpolypeptide substantially non-repetitive. The length of polypeptidewithin which the non-repetitiveness is measured can vary from 3 aminoacids to about 200 amino acids, about from 6 to about 50 amino acids, orfrom about 9 to about 14 amino acids. “Repetitiveness” used in thecontext of polynucleotide sequences refers to the degree of internalhomology in the sequence such as, for example, the frequency ofidentical nucleotide sequences of a given length. Repetitiveness can,for example, be measured by analyzing the frequency of identicalsequences.

A “vector” is a nucleic acid molecule, preferably self-replicating in anappropriate host, which transfers an inserted nucleic acid molecule intoand/or between host cells. The term includes vectors that functionprimarily for insertion of DNA or RNA into a cell, replication ofvectors that function primarily for the replication of DNA or RNA, andexpression vectors that function for transcription and/or translation ofthe DNA or RNA. Also included are vectors that provide more than one ofthe above functions. An “expression vector” is a polynucleotide which,when introduced into an appropriate host cell, can be transcribed andtranslated into a polypeptide(s). An “expression system” usuallyconnotes a suitable host cell comprised of an expression vector that canfunction to yield a desired expression product.

“Serum degradation resistance,” as applied to a polypeptide, refers tothe ability of the polypeptides to withstand degradation in blood orcomponents thereof, which typically involves proteases in the serum orplasma. The serum degradation resistance can be measured by combiningthe protein with human (or mouse, rat, monkey, as appropriate) serum orplasma, typically for a range of days (e.g. 0.25, 0.5, 1, 2, 4, 8, 16days), typically at about 37° C. The samples for these time points canbe run on a Western blot assay and the protein is detected with anantibody. The antibody can be to a tag in the protein. If the proteinshows a single band on the western, where the protein's size isidentical to that of the injected protein, then no degradation hasoccurred. In this exemplary method, the time point where 50% of theprotein is degraded, as judged by Western blots or equivalenttechniques, is the serum degradation half-life or “serum half-life” ofthe protein.

The term “t_(1/2)” as used herein means the terminal half-lifecalculated as ln(2)_(e1). K_(e1) is the terminal elimination rateconstant calculated by linear regression of the terminal linear portionof the log concentration vs. time curve. Half-life typically refers tothe time required for half the quantity of an administered substancedeposited in a living organism to be metabolized or eliminated by normalbiological processes. The terms “t_(1/2)”, “terminal half-life”,“elimination half-life” and “circulating half-life” are usedinterchangeably herein.

“Apparent molecular weight factor” or “apparent molecular weight” arerelated terms referring to a measure of the relative increase ordecrease in apparent molecular weight exhibited by a particular aminoacid sequence. The apparent molecular weight factor is determined usingsize exclusion chromatography (SEC) and similar methods by comparing toglobular protein standards and is measured in “apparent kD” units. Theapparent molecular weight factor is the ratio between the apparentmolecular weight factor and the actual molecular weight; the latterpredicted by adding, based on amino acid composition, the calculatedmolecular weight of each type of amino acid in the composition.

The “hydrodynamic radius” or “Stokes radius” is the effective radius(R_(h) in nm) of a molecule in a solution measured by assuming that itis a body moving through the solution and resisted by the solution'sviscosity. In the embodiments of the invention, the hydrodynamic radiusmeasurements of the XTEN fusion proteins correlate with the ‘apparentmolecular weight factor’, which is a more intuitive measure. The“hydrodynamic radius” of a protein affects its rate of diffusion inaqueous solution as well as its ability to migrate in gels ofmacromolecules. The hydrodynamic radius of a protein is determined byits molecular weight as well as by its structure, including shape andcompactness. Methods for determining the hydrodynamic radius are wellknown in the art, such as by the use of size exclusion chromatography(SEC), as described in U.S. Pat. Nos. 6,406,632 and 7,294,513. Mostproteins have globular structure, which is the most compactthree-dimensional structure a protein can have with the smallesthydrodynamic radius. Some proteins adopt a random and open,unstructured, or ‘linear’ conformation and as a result have a muchlarger hydrodynamic radius compared to typical globular proteins ofsimilar molecular weight.

“Physiological conditions” refer to a set of conditions in a living hostas well as in vitro conditions, including temperature, saltconcentration, pH, that mimic those conditions of a living subject. Ahost of physiologically relevant conditions for use in in vitro assayshave been established. Generally, a physiological buffer contains aphysiological concentration of salt and is adjusted to a neutral pHranging from about 6.5 to about 7.8, and preferably from about 7.0 toabout 7.5. A variety of physiological buffers is listed in Sambrook etal. (1989). Physiologically relevant temperature ranges from about 25°C. to about 38° C., and preferably from about 35° C. to about 37° C.

A “reactive group” is a chemical structure that can be coupled to asecond reactive group. Examples for reactive groups are amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups, aldehyde groups,azide groups. Some reactive groups can be activated to facilitatecoupling with a second reactive group. Non-limiting examples foractivation are the reaction of a carboxyl group with carbodiimide, theconversion of a carboxyl group into an activated ester, or theconversion of a carboxyl group into an azide function.

“Controlled release agent”, “slow release agent”, “depot formulation” or“sustained release agent” are used interchangeably to refer to an agentcapable of extending the duration of release of a polypeptide of theinvention relative to the duration of release when the polypeptide isadministered in the absence of agent. Different embodiments of thepresent invention may have different release rates, resulting indifferent therapeutic amounts.

The terms “antigen”, “target antigen” or “immunogen” are usedinterchangeably herein to refer to the structure or binding determinantthat an antibody fragment or an antibody fragment-based therapeuticbinds to or has specificity against.

The terms “specific binding” or “specifically bind” are usedinterchangeably herein to refer to the high degree of binding affinityof a targeting moiety or binding fusion protein to its correspondingtarget. Typically, specific binding as measured by one or more of theassays disclosed herein would have a dissociation constant or K_(d) ofless than about 10⁻⁶M.

The term “payload” as used herein refers to a protein or peptidesequence that has biological or therapeutic activity; the counterpart tothe pharmacophore of small molecules. Examples of payloads include, butare not limited to, cytokines, enzymes, hormones and blood and growthfactors. Payloads can further comprise genetically fused or chemicallyconjugated moieties such as chemotherapeutic agents, antiviralcompounds, toxins, or contrast agents. These conjugated moieties can bejoined to the rest of the polypeptide via a linker which may becleavable or non-cleavable.

The term “antagonist”, as used herein, includes any molecule thatpartially or fully blocks, inhibits, or neutralizes a biologicalactivity of a native polypeptide disclosed herein. Methods foridentifying antagonists of a polypeptide may comprise contacting anative polypeptide with a candidate antagonist molecule and measuring adetectable change in one or more biological activities normallyassociated with the native polypeptide. In the context of the presentinvention, antagonists may include proteins, nucleic acids,carbohydrates, antibodies or any other molecules that decrease theeffect of a biologically active protein.

The term “agonist” is used in the broadest sense and includes anymolecule that mimics a biological activity of a native polypeptidedisclosed herein. Suitable agonist molecules specifically includeagonist antibodies or antibody fragments, fragments or amino acidsequence variants of native polypeptides, peptides, small organicmolecules, etc. Methods for identifying agonists of a native polypeptidemay comprise contacting a native polypeptide with a candidate agonistmolecule and measuring a detectable change in one or more biologicalactivities normally associated with the native polypeptide.

“Activity” for the purposes herein refers to an action or effect of acomponent of a fusion protein consistent with that of the correspondingnative biologically active protein, wherein “biological activity” refersto an in vitro or in vivo biological function or effect, including butnot limited to receptor binding, antagonist activity, agonist activity,or a cellular or physiologic response.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” is used interchangeably herein. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the subject, notwithstanding that thesubject may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a subjectat risk of developing a particular disease, or to a subject reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made.

A “therapeutic effect”, as used herein, refers to a physiologic effect,including but not limited to the cure, mitigation, amelioration, orprevention of disease in humans or other animals, or to otherwiseenhance physical or mental wellbeing of humans or animals, caused by afusion polypeptide of the invention other than the ability to induce theproduction of an antibody against an antigenic epitope possessed by thebiologically active protein. Determination of a therapeuticallyeffective amount is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

The terms “therapeutically effective amount” and “therapeuticallyeffective dose”, as used herein, refers to an amount of a biologicallyactive protein, either alone or as a part of a fusion proteincomposition, that is capable of having any detectable, beneficial effecton any symptom, aspect, measured parameter or characteristics of adisease state or condition when administered in one or repeated doses toa subject. Such effect need not be absolute to be beneficial.

The term “therapeutically effective dose regimen”, as used herein,refers to a schedule for consecutively administered doses of abiologically active protein, either alone or as a part of a fusionprotein composition, wherein the doses are given in therapeuticallyeffective amounts to result in sustained beneficial effect on anysymptom, aspect, measured parameter or characteristics of a diseasestate or condition.

I). General Techniques

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook, J.et al., “Molecular Cloning: A Laboratory Manual,” 3^(rd) edition, ColdSpring Harbor Laboratory Press, 2001; “Current protocols in molecularbiology”, F. M. Ausubel, et al. eds., 1987; the series “Methods inEnzymology,” Academic Press, San Diego, Calif.; “PCR 2: a practicalapproach”, M. J. MacPherson, B. D. Hames and G. R. Taylor eds., OxfordUniversity Press, 1995; “Antibodies, a laboratory manual” Harlow, E. andLane, D. eds., Cold Spring Harbor Laboratory, 1988; “Goodman & Gilman'sThe Pharmacological Basis of Therapeutics,” 11^(th) Edition,McGraw-Hill, 2005; and Freshney, R. I., “Culture of Animal Cells: AManual of Basic Technique,” 4^(th) edition, John Wiley & Sons, Somerset,N.J., 2000, the contents of which are incorporated in their entiretyherein by reference.

II). Binding Fusion Protein Compositions

(a) The present invention relates, in part, to binding fusion protein(“BFP”) compositions comprising fusion proteins of polypeptide targetingmoieties linked to one or more extended recombinant polypeptides(“XTEN”). In particular, the invention provides isolated binding fusionprotein compositions useful in the treatment of diseases, disorders orconditions in which the targeting moiety can be directed to an antigen,ligand, or receptor implicated in, associated with, or that modulates adisease, disorder or condition, while the XTEN carrier portion can bedesigned to confer a desired half-life or enhanced pharmaceuticalproperty on the binding fusion protein, as described more fully below.The binding fusion proteins of the present invention may act as agonistsor antagonists. In one embodiment, the composition can further comprisea second targeting moiety or multiple targeting moieties that can havebinding affinity for the same or a different target, resulting inbispecific or multivalent binding fusion proteins. The inventionprovides several different forms and configurations of targetingmoieties and XTEN. The binding fusion proteins of the embodimentsdisclosed herein exhibit one or more or any combination of theproperties and/or the embodiments as detailed herein.

Targets

In general, the targeting moieties of the subject binding fusion proteincompositions exhibit a binding specificity to a given target or anotherdesired biological characteristic when used in vivo or when utilized inan in vitro assay. The subject binding fusion proteins comprising two ormore targeting moieties can be designed to bind the same target,different epitopes on the same target, or different targets by theselective incorporation of targeting moieties with binding affinity tothe respective binding sites.

The targets to which the targeting moieties of the subject bindingfusion protein compositions can be directed include cytokines,cytokine-related proteins, cytokine receptors, chemokines, chemokinesreceptors, cell surface receptors or antigens, hormones or similarcirculating proteins or peptides, oligonucleotides, or enzymaticsubstrates. The targets are generally associated with a disease,disorder or condition. As used herein, “a target associated with adisease, disorder or condition” means that the target is eitherexpressed or overexpressed by disease cells or tissues, the targetcauses or is a mediator or is a by-product of the disease, disorder orcondition, or the target is generally found in higher concentrations ina subject with the disease, disorder or condition, or the target isfound in higher than baseline concentrations within or proximal to theareas of the disease, disorder or condition in the subject. Anon-limiting example of the foregoing is the target HER2, which isimplicated in approximately 30 percent of breast cancers due to anamplification of the HER2/neu gene or over-expression of its proteinproduct. Over-expression of the HER2 receptor in breast cancer isassociated with increased disease recurrence and worse prognosis, and ahumanized anti-Her2/neu antibody is used in treatment of breast cancersexpressing the HER2 receptor (see for example U.S. Pat. No. 4,753,894).

In one embodiment, the one or more targeting moieties can have bindingaffinity to targets selected from, but not limited to the targets ofTable 1.

TABLE 1 Targets for targeting moieties Target ABCF1; ACVR1; ACVR1B;ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AIF1; AIG1;AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC;APOC1; APRIL; AR; AZGP1 (zinc-a-glycoprotein); A4 integrin; B7; B7.1;B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS;BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2;BPAG1 (plectin); BRCA1; C19orf10 (IL27w); C3; C4A; C5; C5R1; CANT1;CASP1; CASP4; CAV1; CCBP2 (D6/JAB61); CCL1 (1-309); CCL11 (eotaxin);CCL13 (MCP-4); CCL15 (MIP-1d); CCL16 (HCC-4); CCL17 (TARC); CCL18(PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21(MIP-2); SLC; exodus- 2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24(MPIF-2/eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC);CCL28; CCL3 (MIP-1a); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3); CCL8(mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1/HM145); CCR2(mcp-1RB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR13); CCR6(CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBI1); CCR8(CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR);CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38;CD3E; CD3G; CD3Z; CD4; CD11a (LFA-1 integrin alphaL); CD40; CD40L; CD44;CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83;CD86; CD340; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19;CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9;CDKN1A (p21Wap1/Cip1); CDKN1B (p27Kip1); CDKN1C; CDKN2A (p16INK4a);CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; Chitinase; CHST10;CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3;CLDN7 (claudin-7); CLN3; CLU (clusterin); cMET; CMKLR1; CMKOR1 (RDC1);CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2(GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB (cathepsin B);CX3CL1 (SCYD1); CX3CR1 (V28); CXCL1 (GRO1); CXCL10(IP- 10); CXCL11(I-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3(GRO3); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP;DES; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNA1; EFNA3; EFNB2;EGF; EGFR; ELAC2; elastase; ENG; ENO1; ENO2; ENO3; EPHB4; EPO; ERBB-2(Her2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A;FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13;FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22;FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8;FGF9; FGFR3; FIGF (VEGFD); FIL1 (EPSILON); FIL1 (ZETA); FLJ12584;FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1 (FRA-1); FY (DARC);GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1;GM-CSF; GNAS1; GNRH1; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10(C10); GRP; GSN (Gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9;HER2; HGF; HIF1A; HIP1; histamine and histamine receptors; HLA-A;HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFNA1;IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; IFNW1; IGBP1; IGF1;IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11;IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2;IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP;IL18R1; IL18RAP; IL19; IL1A; IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8;IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2;IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25;IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA;IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); IL7; IL7R;IL8; IL8RA; IL8RB; IL8RB; IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4;IRAK1; IRAK2; ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3;ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG;KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4;KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair-specifictype II keratin); LAMA5; LEP (leptin); LFA3; LIGHT; Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAGor Omgp; MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF; MIP-2; MKI67 (Ki-67);MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSS1; MUC1(mucin); MYC; MYD88; NCK2; neurocan; NFKB1; NFKB2; NGFB (NGF); NGFR;NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5;NPPB; NROB1; NROB2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NRII2; NRII3;NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1;NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1;P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4(CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDC1;PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA;PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RANKL; RARB; RGS1;RGS13; RGS3; RNF110 (ZNF144); ROBO2; RSV; SI00A2; SCGB1D2 (lipophilinB); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelialMonocyte-activating cytokine); SDF2; SERPINA1; SERPINA3; SERPINB5(maspin); SERPINE1 (PAI-1); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1;SLC43A1; SLIT2; SPP1; SPRR1B (Spr1); ST6GAL1; STAB1; STAT6; STEAP;STEAP2; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGFB111; TGFB2;TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1);THBS2; THBS4; THPO; TIE (Tie-1); TIMP3; tissue factor; TLR10; TLR2;TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNFAIP2 (B94);TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6(Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE);TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15(VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6(FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BBligand); TOLLIP; Toll-like receptors (TLR1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12 to TLR-13); TOP2A (topoisomerase Iia); TP53; TPM1; TPM2; TRADD;TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP;TWEAK; VAP1; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-1; VLA-4; XCL1(lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5/CCXCR1); YY1; ZFPM2.

In one embodiment, the one or more targeting moieties can have bindingaffinity to one or more tumor-associated antigens (TAA) known to beexpressed on tumor or cancer cells or are otherwise associated withtumors or cancers. Tumor-associated antigens are known in the art, andare generally regarded as effective cellular targets for cancerdiagnosis and therapy. In particular, researchers have sought toidentify TAA that are specifically expressed on the surface of one ormore particular types of cancer cell as compared to on one or morenormal non-cancerous cells, and has given rise to the ability tospecifically target cancer cells for destruction via antibody-basedtherapies. Non-limiting examples of TAA are listed in Table 2. In oneembodiment, a binding fusion protein comprises a targeting moiety withbinding affinity to a TAA target selected from Table 2. In anotherembodiment, the binding fusion protein comprises two or more targetingmoieties that have binding affinity to one or more TAA targets selectedfrom the targets of Table 2.

TABLE 2 Tumor-associated antigen targets TAA targets (synonyms)Accession Number and References Her2 (ErbB2) GenBank accession no.M11730; U.S. Pat. No. 5,869,445; WO2004048938; WO2004027049;WO2004009622; WO2003081210; WO2003089904; WO2003016475; US2003118592;WO2003008537; WO2003055439; WO2003025228; WO200222636; WO200212341;WO200213847; WO200214503; WO200153463; WO200141787; WO200044899;WO200020579; WO9630514; EP1439393; WO2004043361; WO2004022709;WO200100244 BMPR1B (bone morphogenetic protein GenBank accession no.NM_001203; WO2004063362; receptor-type IB) WO2003042661; US 2003134790;WO2002102235; WO2003055443; WO200299122; WO2003029421; WO2003024392;WO200298358' WO200254940; WO200259377; WO200230268 E16 (LAT1, SLC7A5)GenBank accession no. NM_003486); WO2004048938; WO2004032842;WO2003042661; WO2003016475; WO200278524; WO200299074; WO200286443;WO2003003906; WO200264798; WO200014228; US2003224454; WO2003025138STEAP1 (six transmembrane epithelial GenBank accession no. NM_012449;WO2004065577; antigen of prostate) WO2004027049; EP1394274;WO2004016225; WO2003042661; US2003157089; US2003185830; US2003064397;WO200289747618; WO2003022995 STEAP2 (six transmembrane epithelialGenBank accession no. AF455138; WO2003087306; antigen of prostate 2)US2003064397; WO200272596; WO200172962; WO2003104270; WO2003104270;US2004005598; WO2003042661; US2003060612; WO200226822; WO200216429CA125/0772P (MUC16) GenBank accession no. AF361486; WO2004045553;WO200292836; WO200283866; US2003124140 megakaryocyte potentiating factorGenBank accession no. NM_005823; WO2003101283; (MPF, mesothelin)WO2002102235; WO2002101075; WO200271928; WO9410312 Na/Pi cotransportertype IIb (NaPi3b) GenBank accession no. NM_006424; WO2004022778;EP1394274; WO2002102235; EP875569; WO200157188; WO2004032842;WO200175177 Semaphorin 5b (SEMA5B, SEMAG) GenBank accession no.AB040878; WO2004000997; WO2003003984; WO200206339; WO200188133;WO2003054152; WO2003101400 Prostate cancer stem cell antigen GenBankaccession no. AY358628; US2003129192; (PSCA hlg) US2004044180;US2004044179; US2003096961; US2003232056; WO2003105758; US2003206918;EP1347046; WO2003025148 ETBR (Endothelin type B receptor) GenBankaccession no. AY275463; WO2004045516; WO2004048938; WO2004040000;WO2003087768; WO2003016475; WO2003016475; WO200261087; WO2003016494;WO2003025138; WO200198351; EP522868; WO200177172; US2003109676; U.S.Pat. No. 6,518,404; U.S. Pat. No. 5,773,223; WO2004001004 TRPV4(Transient receptor potential U.S. patent application No. 20090208514cation channel, subfamily V) CDC45L GenBank Accession NO. AJ223728; U.S.patent application No. 20090208514 CRIPTO (CR, CR1, CRGF) GenBankaccession no. NP_003203 or NM_003212; US2003224411; WO2003083041;WO2003034984; WO200288170; WO2003024392; WO200216413; WO200222808; U.S.Pat. No. 5,854,399; U.S. Pat. No. 5,792,616 CD21 (CR2 (Complementreceptor 2) GenBank accession no. M26004; WO2004045520; or C3DR(C3d/Epstein Barr virus US2004005538; WO2003062401; WO2004045520;receptor) WO9102536; WO2004020595 CD79b (CD79B, CD79β, IGb GenBankaccession no. NM_000626 or 11038674; (immunoglobulin-associated beta),WO2004016225; WO2003087768; US2004101874; B29) WO2003062401;WO200278524; US2002150573; U.S. Pat. No. 5,644,033; WO2003048202; WO99/558658, U.S. Pat. No. 6,534,482; WO200055351 FcRH2 (IFGP4, IRTA4,SPAP1A GenBank accession no. NM_030764, AY358130; (SH2 domain containingphosphatase WO2004016225; WO2003077836; WO200138490; anchor protein 1a),SPAP1B, SPAP1C) WO2003097803; WO2003089624 NCA (CEACAM6) GenBankaccession no. M18728; WO2004063709; EP1439393; WO2004044178;WO2004031238; WO2003042661; WO200278524; WO200286443; WO200260317 MDP(DPEP1) GenBank accession no. BC017023; WO2003016475; WO200264798 IL20Rα(IL20Ra, ZCYTOR7) GenBank accession no. AF184971; EP1394274;US2004005320; WO2003029262; WO2003002717; WO200222153; US2002042366;WO200146261; WO200146232; WO9837193 BECAN (Brevican core protein)GenBank accession no. AF229053; US2003186372; US2003186373;US2003119131; US2003119122; US2003119126; US2003119121; US2003119129;US2003119130; US2003119128; US2003119125; WO2003016475; WO200202634EphB2R (DRT, ERK, Hek5, EPHT3, GenBank accession no. NM_004442;WO2003042661; Tyro5) WO200053216; WO2004065576 (Claim 1); WO2004020583;WO2003004529; WO200053216 B7h (ASLG659) GenBank accession no. AX092328;US20040101899; WO2003104399; WO2004000221; US2003165504; US2003124140;US2003065143; WO2002102235; US2003091580; WO200210187; WO200194641;WO200202624; US2002034749; WO200206317; WO200271928; WO200202587;WO200140269; WO200036107; WO2004053079; WO2003004989; WO200271928 PSCA(Prostate stem cell antigen GenBank accession no. AJ297436;WO2004022709; precursor EP1394274; US2004018553; WO2003008537 (Claim 1);WO200281646; WO2003003906; WO200140309; US2001055751; WO200032752;WO9851805; WO9851824; WO9840403 BAFF-R (B cell-activating factor GenBankaccession No. AF116456; WO2004058309; receptor, BLyS receptor 3, BR3)WO2004011611; WO2003045422; WO2003014294; WO2003035846; WO200294852;WO200238766; WO200224909 CD22 (B-cell receptor CD22-β-form, GenBankaccession No. AK026467; WO2003072036 BL-CAM, Lyb-8, Lyb8, SIGLEC-2,FLJ22814) CD79a (immunoglobulin-associated GenBank accession No.NP_001774.10; WO2003088808, alpha) US20030228319; WO2003062401;US2002150573; WO9958658; WO9207574; U.S. Pat. No. 5,644,033 CXCR5(Burkitt's lymphoma GenBank accession No. NP_001707.1; WO2004040000;receptor 1) WO2004015426; US2003105292; U.S. Pat. No. 6,555,339;WO200261087; WO200157188; WO200172830; WO200022129; WO9928468; U.S. Pat.No. 5,440,021; WO9428931; WO9217497 HLA-DOB GenBank accession No.NP_002111.1; WO9958658; U.S. Pat. No. 6,153,408; U.S. Pat. No.5,976,551; U.S. Pat. No. 6,011,146 P2X5 GenBank accession No.NP_002552.2; WO2004047749; WO2003072035; WO200222660; WO2003093444;WO2003087768; WO2003029277 CD72 (B-cell differentiation antigen GenBankaccession No. NP_001773.1; WO2004042346; CD72, Lyb-2) WO2003026493;WO200075655 CD180 (LY64) GenBank accession No. NP_005573.1;US2002193567; WO9707198; WO2003083047; WO9744452 FcRH1 (Fc receptor-likeprotein 1) GenBank accession No. NP_443170.1) WO2003077836; WO200138490;WO2003089624; EP1347046; WO2003089624 IRTA2 (Immunoglobulin superfamilyGenBank accession No. Human: AF343662, AF343663, receptor translocationassociated 2) AF343664, AF343665, AF369794, AF397453; WO2003024392;WO2003077836; WO200138490 TENB2 (TMEFF2, tomoregulin, GenBank accessionNo. AF179274; AY358907, CAF85723, TPEF, HPP1) CQ782436; WO2004074320;WO2003042661; WO2003009814; EP1295944; WO200230268; WO200190304;US2004249130; US2004022727; WO2004063355; US2004197325; US2003232350;US2004005563; US2003124579 CS1 (CRACC, 19A, APEX-1, GenBank AccessionNo. NM 021181; US 20100168397 FOAP12) DLL4 GenBank Accession No. NM019074; US 20100303812 Lewis Y ADB235860; U.S. Pat. No. 7,879,983 CD40(Bp50, CDW40, MGC9013, AL035662.65; U.S. Pat. No. 6,946,129 TNFRSF5,p50) OBA1 (5T4) GenBank Accession No. NP_001159864.1; US 20100021483 p97Woodbury et al., 1980, Proc. Natl. Acad. Sci. USA 77: 2183-2186; Brownet al., 1981, J. Immunol. 127: 539-546 carcinoembryonic antigen (CEA)GenBank Accession No. NP_004354.2; U.S. Pat. No. 6,676,924 TAG-72 U.S.Pat. No. 7,256,004 DNA Neuropilin -1 (NRP1) GenBank Accession No.NP_001019799.1; US 20080213268 A33 GenBank Accession No. NP_005805.1;U.S. Pat. No. 7,579,187 Mucin-1 (MUC1) GenBank Accession No.NP_001018016.1; NP_001018017.1; U.S. Pat. No. 7,183,388 ED-B fibronectinU.S. Pat. No. 7,785,591 Thomsen-Friedenreich antigen (TF) U.S. Pat. No.7,374,755; US 20100297159

(b) Extended Recombinant Polypeptides

In one aspect, the invention provides XTEN polypeptide compositions thatare useful as a fusion protein partner to which one or more targetingmoieties can be linked, resulting in a binding fusion protein. XTEN aregenerally extended length polypeptides with non-naturally occurring,substantially non-repetitive sequences that are composed mainly of smallhydrophilic amino acids, with the sequence having a low degree or nosecondary or tertiary structure under physiologic conditions. XTEN haveutility as fusion protein partners in that they serve in various roles,conferring certain desirable pharmacokinetic, physicochemical andpharmaceutical properties, amongst other properties described below,when linked to a targeting moiety to a create a fusion protein.

In some embodiments, XTEN are long polypeptides having greater than 100to about 3000 residues, and preferably 400 to about 3000 residues whenused as a carrier or cumulatively when more than one XTEN unit is usedin a single fusion protein with a targeting moiety; e.g., a linker and acarrier or an N-terminal XTEN and a carrier. In other embodiments,shorter XTEN sequences can be used as linkers to join components of thebinding fusion proteins or to enhance expression as an N-terminal XTEN,as described more fully below.

The selection criteria for the XTEN used to create the inventivecompositions generally relate to attributes of physical/chemicalproperties and conformational structure of the XTEN that can be, inturn, used to confer enhanced pharmaceutical and pharmacokineticproperties to the compositions. The XTEN of the present invention mayexhibit one or more of the following advantageous properties:conformational flexibility, enhanced aqueous solubility, high degree ofprotease resistance, low immunogenicity, low binding to mammalianreceptors, and increased hydrodynamic (or Stokes) radii; properties thatcan make them particularly useful as fusion protein partners andscaffolds for drug conjugates. Non-limiting examples of the propertiesof the inventive compositions that may be enhanced by XTEN includeincreases in the overall solubility and/or metabolic stability, reducedsusceptibility to proteolysis, reduced immunogenicity, reduced rate ofabsorption when administered subcutaneously or intramuscularly, andenhanced pharmacokinetic properties such as longer terminal half-lifeand increased area under the curve (AUC), slower absorption aftersubcutaneous or intramuscular injection (compared to agents not linkedto XTEN administered by a parenteral route) such that the C_(max) islower, which may, in turn, result in reductions in adverse effects that,collectively, can result in an increased period of time that a fusionprotein composition administered to a subject retains therapeuticactivity.

A variety of methods and assays are known in the art for determining thephysical/chemical properties of proteins such as the compositionscomprising the inventive XTEN; properties such as secondary or tertiarystructure, solubility, protein aggregation, melting properties,contamination and water content. Such methods include analyticalcentrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion,HPLC-reverse phase, light scattering, capillary electrophoresis,circular dichroism, differential scanning calorimetry, fluorescence,HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy,refractometry, and UV/Visible spectroscopy. Additional methods aredisclosed in Arnau et al, Prot Expr and Purif (2006) 48, 1-13.Application of these methods to the invention would be within the graspof a person skilled in the art.

In one embodiment, XTEN are designed to behave like denatured peptidesequences under physiological conditions, despite the extended length ofthe polymer. Denatured describes the state of a peptide in solution thatis characterized by a large conformational freedom of the peptidebackbone. Most peptides and proteins adopt a denatured conformation inthe presence of high concentrations of denaturants or at elevatedtemperature. Peptides in denatured conformation have, for example,characteristic circular dichroism (CD) spectra and are characterized bya lack of long-range interactions as determined by NMR. “Denaturedconformation” and “unstructured conformation” are used synonymouslyherein. In one embodiment, the invention provides XTEN sequences that,under physiologic conditions, can resemble denatured sequences largelydevoid in secondary structure. In one embodiment, the XTEN sequences canbe substantially devoid of secondary structure under physiologicconditions. “Largely devoid,” as used in this context, means that lessthan 50% of the XTEN amino acid residues of the XTEN sequence contributeto secondary structure as measured or determined by the means describedherein. “Substantially devoid,” as used in this context, means that atleast about 60%, or about 70%, or about 80%, or about 90%, or about 95%,or at least about 99% of the XTEN amino acid residues of the XTENsequence do not contribute to secondary structure, as measured ordetermined by the means described herein.

A variety of methods have been established in the art to discern thepresence or absence of secondary and tertiary structures in a givenpolypeptide. In particular, secondary structure can be measuredspectrophotometrically, e.g., by circular dichroism spectroscopy in the“far-UV” spectral region (190-250 nm). Secondary structure elements,such as alpha-helix and beta-sheet, each give rise to a characteristicshape and magnitude of CD spectra. Secondary structure can also bepredicted for a polypeptide sequence via certain computer programs oralgorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y.,et al. (1974) Biochemistry, 13: 222-45) and theGarnier-Osguthorpe-Robson (“GOR”) algorithm (Garnier J, Gibrat J F,Robson B. (1996), GOR method for predicting protein secondary structurefrom amino acid sequence. Methods Enzymol 266:540-553), as described inUS Patent Application Publication No. 20030228309A1. For a givensequence, the algorithms can predict whether there exists some or nosecondary structure at all, expressed as the total and/or percentage ofresidues of the sequence that form, for example, alpha-helices orbeta-sheets or the percentage of residues of the sequence predicted toresult in random coil formation (which lacks secondary structure).

In one embodiment, the XTEN sequences used in the subject fusion proteincompositions have an alpha-helix percentage ranging from 0% to less thanabout 5% as determined by the Chou-Fasman algorithm. In anotherembodiment, the XTEN sequences of the fusion protein compositions have abeta-sheet percentage ranging from 0% to less than about 5% asdetermined by the Chou-Fasman algorithm. In some embodiments, the XTENsequences of the fusion protein compositions have an alpha-helixpercentage ranging from 0% to less than about 5% and a beta-sheetpercentage ranging from 0% to less than about 5% as determined by theChou-Fasman algorithm. In one embodiment, the XTEN sequences of thefusion protein compositions have an alpha-helix percentage less thanabout 2% and a beta-sheet percentage less than about 2%. The XTENsequences of the fusion protein compositions have a high degree ofrandom coil percentage, as determined by the GOR algorithm. In someembodiments, an XTEN sequence have at least about 80%, more preferablyat least about 90%, more preferably at least about 91%, more preferablyat least about 92%, more preferably at least about 93%, more preferablyat least about 94%, more preferably at least about 95%, more preferablyat least about 96%, more preferably at least about 97%, more preferablyat least about 98%, and most preferably at least about 99% random coil,as determined by the GOR algorithm. In one embodiment, the XTENsequences of the fusion protein compositions have an alpha-helixpercentage ranging from 0% to less than about 5% and a beta-sheetpercentage ranging from 0% to less than about 5% as determined by theChou-Fasman algorithm and at least about 90% random coil, as determinedby the GOR algorithm. In another embodiment, the XTEN sequences of thefusion protein compositions have an alpha-helix percentage less thanabout 2% and a beta-sheet percentage less than about 2% at least about90% random coil, as determined by the GOR algorithm.

1. Non-Repetitive Sequences

It is specifically contemplated that the XTEN sequences of the bindingfusion protein embodiments are substantially non-repetitive. In general,repetitive amino acid sequences have a tendency to aggregate or formhigher order structures, as exemplified by natural repetitive sequencessuch as collagens and leucine zippers. These repetitive amino acids mayalso tend to form contacts resulting in crystalline or pseudocrystalinestructures. In contrast, the low tendency of non-repetitive sequences toaggregate enables the design of long-sequence XTENs with a relativelylow frequency of charged amino acids that would otherwise be likely toaggregate if the sequences were repetitive. In one embodiment, the XTENsequences have greater than about 36 to about 1000 amino acid residues,or greater than about 100 to about 3000 amino acid residues in which nothree contiguous amino acids in the sequence are identical amino acidtypes unless the amino acid is serine, in which case no more than threecontiguous amino acids are serine residues. In the foregoing embodiment,the XTEN sequence is “substantially non-repetitive.” In anotherembodiment, as described more fully below, the XTEN sequences of thecompositions comprise non-overlapping sequence motifs of 9 to 14 aminoacid residues wherein the motifs consist of 4 to 6 types of amino acidsselected from glycine (G), alanine (A), serine (S), threonine (T),glutamate (E) and proline (P), and wherein the sequence of any twocontiguous amino acid residues in any one motif is not repeated morethan twice in the sequence motif. In the foregoing embodiment, the XTENsequence is “substantially non-repetitive.”

The degree of repetitiveness of a polypeptide or a gene can be measuredby computer programs or algorithms or by other means known in the art.According to the current invention, algorithms to be used in calculatingthe degree of repetitiveness of a particular polypeptide, such as anXTEN, are disclosed herein, and examples of sequences analyzed byalgorithms are provided (see Examples, below). In one embodiment, therepetitiveness of a polypeptide of a predetermined length can becalculated (hereinafter “subsequence score”) according to the formulagiven by Equation I:

$\begin{matrix}{{{Subsequence}\mspace{14mu}{score}} = \frac{\sum\limits_{i = 1}^{m}{Count}_{i}}{m}} & I\end{matrix}$

-   -   wherein: m=(amino acid length of polypeptide)−(amino acid length        of subsequence)+1; and        -   Count_(i)=cumulative number of occurrences of each unique            subsequence within sequence_(i)

An algorithm termed “SegScore” was developed to apply the foregoingequation to quantitate repetitiveness of polypeptides, such as an XTEN,providing the subsequence score wherein sequences of a predeterminedamino acid length are analyzed for repetitiveness by determining thenumber of times (a “count”) a unique subsequence of length “s” appearsin the set length, divided by the absolute number of subsequences withinthe predetermined length of the sequence. FIG. 37 depicts a logicflowchart of the SegScore algorithm, while FIG. 38 portrays a schematicof how a subsequence score is derived for a fictitious XTEN with 11amino acids and a subsequence length of 3 amino acid residues. Forexample, a predetermined polypeptide length of 200 amino acid residueshas 192 overlapping 9-amino acid subsequences and 198 3-mersubsequences, but the subsequence score of any given polypeptide willdepend on the absolute number of unique subsequences and how frequentlyeach unique subsequence (meaning a different amino acid sequence)appears in the predetermined length of the sequence. In the context ofthe present invention wherein the algorithm is used to determine thedegree of repetitiveness in a polypeptide, the variable “amino acidlength of polypeptide” is set to 200 amino acids and the variable “aminoacid length of subsequence” is set to 3 amino acids. Thus, thesubsequence score will equal the sum of occurrences of each unique 3-merframe across a 200 consecutive amino acid sequence of the polypeptidedivided by the absolute number of unique 3-mer subsequences within the200 amino acid sequence. Examples of such subsequence scores derivedfrom the first 200 amino acids of repetitive and non-repetitivepolypeptides are presented in Example 58.

In one embodiment, the present invention provides binding fusionproteins comprising one XTEN in which the XTEN has a subsequence scoreof less than 10, or less than 9, or less than 8, or less than 7, or lessthan 6, or less than 5, or less. In another embodiment, the inventionprovides binding fusion proteins comprising two more XTEN m which atleast one XTEN has a subsequence score of less than 10, or less than 9,or less than 8, or less than 7, or less than 6, or less than 5, or less.In yet another embodiment, the invention provides binding fusionproteins comprising at least two XTEN in which each individual XTEN of36 or more amino acids has a subsequence score of less than 10, or lessthan 9, or less than 8, or less than 7, or less than 6, or less than 5,or less. In the embodiments of this paragraph, the XTEN is characterizedas “substantially non-repetitive.”.

It is believed that the non-repetitive characteristic of XTEN of thepresent invention contributes to many of the enhanced physicochemicaland biological properties of the binding fusion proteins; either solelyor in conjunction with the choice of the particular types of amino acidsthat predominate in the XTEN of the compositions disclosed herein. Theseproperties include a higher degree of expression of the fusion proteinin the host cell, greater genetic stability of the gene encoding XTEN,and a greater degree of solubility and less tendency to aggregate of theresulting binding fusion proteins compared to fusion proteins comprisingpolypeptides having repetitive sequences. These properties permit moreefficient manufacturing, lower cost of goods, and facilitate theformulation of XTEN-comprising pharmaceutical preparations containingextremely high drug concentrations, in some cases exceeding 100 mg/ml.Furthermore, the XTEN polypeptide sequences of the embodiments aredesigned to have a low degree of internal repetitiveness in order toreduce or substantially eliminate immunogenicity when administered to amammal Polypeptide sequences composed of short, repeated motifs largelylimited to only three amino acids, such as glycine, serine andglutamate, may result in relatively high antibody titers whenadministered to a mammal despite the absence of predicted T-cellepitopes in these sequences. This may be caused by the repetitive natureof polypeptides, as it has been shown that immunogens with repeatedepitopes, including protein aggregates, cross-linked immunogens, andrepetitive carbohydrates are highly immunogenic and can, for example,result in the cross-linking of B-cell receptors causing B-cellactivation. (Johansson, J., et al. (2007) Vaccine, 25:1676-82; Yankai,Z., et al. (2006) Biochem Biophys Res Commun, 345:1365-71; Hsu, C. T.,et al. (2000) Cancer Res, 60:3701-5); Bachmann M F, et al. Eur JImmunol. (1995) 25(12):3445-3451).

2. Exemplary Sequence Motifs

The present invention encompasses XTEN used as fusion partners thatcomprise multiple units of shorter sequences, or motifs, in which theamino acid sequences of the motifs are non-repetitive. Thenon-repetitive property is met despite the use of a “building block”approach using a library of sequence motifs that are multimerized tocreate the XTEN sequences. Thus, while an XTEN sequence may consist ofmultiple units of as few as four different types of sequence motifs,because the motifs themselves generally consist of non-repetitive aminoacid sequences, the overall XTEN sequence is designed to render thesequence substantially non-repetitive.

In one embodiment, XTEN have a non-repetitive sequence of greater thanabout 36 to about 3000 amino acid residues wherein at least about 80%,or at least about 85%, or at least about 90%, or at least about 95%, orat least about 97%, or about 100% of the XTEN sequence consists ofnon-overlapping sequence motifs, wherein each of the motifs has about 9to 36 amino acid residues. In other embodiments, at least about 80%, orat least about 85%, or at least about 90%, or at least about 95%, or atleast about 97%, or about 100% of the XTEN sequence consists ofnon-overlapping sequence motifs wherein each of the motifs has 9 to 14amino acid residues. In still other embodiments, at least about 80%, orat least about 85%, or at least about 90%, or at least about 95%, or atleast about 97%, or about 100% of the XTEN sequence component consistsof non-overlapping sequence motifs wherein each of the motifs has 12amino acid residues. In these embodiments, it is preferred that thesequence motifs be composed mainly or exclusively of small hydrophilicamino acids, such that the overall sequence has an unstructured,flexible characteristic. Examples of amino acids that are included inXTEN are, e.g., arginine, lysine, threonine, alanine, asparagine,glutamine, aspartate, glutamate, serine, and glycine. As a result oftesting variables such as codon optimization, assembly polynucleotidesencoding sequence motifs, expression of protein, charge distribution andsolubility of expressed protein, and secondary and tertiary structure,it was discovered that XTEN compositions with enhanced characteristicsmainly include glycine (G), alanine (A), serine (S), threonine (T),glutamate (E) and proline (P) residues wherein the sequences aredesigned to be substantially non-repetitive. In one embodiment, XTENsequences have predominately four to six types of amino acids selectedfrom glycine (G), alanine (A), serine (S), threonine (T), glutamate (E)or proline (P) that are arranged in a substantially non-repetitivesequence that is greater than about 36 to about 3000 amino acid residuesin length. In some embodiments, XTEN have sequences of greater thanabout 36 to about 3000 amino acid residues wherein at least about 80% ofthe sequence consists of non-overlapping sequence motifs wherein each ofthe motifs has 9 to 36 amino acid residues wherein each of the motifsconsists of 4 to 6 types of amino acids selected from glycine (G),alanine (A), serine (S), threonine (T), glutamate (E) and proline (P),and wherein the content of any one amino acid type in the full-lengthXTEN does not exceed 30%. In other embodiments, at least about 90% ofthe XTEN sequence consists of non-overlapping sequence motifs whereineach of the motifs has 9 to 36 amino acid residues wherein the motifsconsist of 4 to 6 types of amino acids selected from glycine (G),alanine (A), serine (S), threonine (T), glutamate (E) and proline (P),and wherein the content of any one amino acid type in the full-lengthXTEN does not exceed 30%. In other embodiments, at least about 90% ofthe XTEN sequence consists of non-overlapping sequence motifs whereineach of the motifs has 12 amino acid residues consisting of 4 to 6 typesof amino acids selected from glycine (G), alanine (A), serine (S),threonine (T), glutamate (E) and proline (P), and wherein the content ofany one amino acid type in the full-length XTEN does not exceed 30%. Inyet other embodiments, at least about 90%, or about 91%, or about 92%,or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, orabout 98%, or about 99%, to about 100% of the XTEN sequence consists ofnon-overlapping sequence motifs wherein each of the motifs has 12 aminoacid residues consisting of glycine (G), alanine (A), serine (S),threonine (T), glutamate (E) and proline (P), and wherein the content ofany one amino acid type in the full-length XTEN does not exceed 30%.

In still other embodiments, XTENs comprise non-repetitive sequences ofgreater than about 36 to about 3000 amino acid residues wherein at leastabout 80%, or at least about 90%, or about 91%, or about 92%, or about93%, or about 94%, or about 95%, or about 96%, or about 97%, or about98%, or about 99% of the sequence consists of non-overlapping sequencemotifs of 9 to 14 amino acid residues wherein the motifs consist of 4 to6 types of amino acids selected from glycine (G), alanine (A), serine(S), threonine (T), glutamate (E) and proline (P), and wherein thesequence of any two contiguous amino acid residues in any one motif isnot repeated more than twice in the sequence motif. In otherembodiments, at least about 90%, or about 91%, or about 92%, or about93%, or about 94%, or about 95%, or about 96%, or about 97%, or about98%, or about 99% of an XTEN sequence consists of non-overlappingsequence motifs of 12 amino acid residues wherein the motifs consist offour to six types of amino acids selected from glycine (G), alanine (A),serine (S), threonine (T), glutamate (E) and proline (P), and whereinthe sequence of any two contiguous amino acid residues in any onesequence motif is not repeated more than twice in the sequence motif. Inother embodiments, at least about 90%, or about 91%, or about 92%, orabout 93%, or about 94%, or about 95%, or about 96%, or about 97%, orabout 98%, or about 99% of an XTEN sequence consists of non-overlappingsequence motifs of 12 amino acid residues wherein the motifs consist ofglycine (G), alanine (A), serine (S), threonine (T), glutamate (E) andproline (P), and wherein the sequence of any two contiguous amino acidresidues in any one sequence motif is not repeated more than twice inthe sequence motif. In yet other embodiments, XTENs consist of 12 aminoacid sequence motifs wherein the amino acids are selected from glycine(G), alanine (A), serine (S), threonine (T), glutamate (E) and proline(P), and wherein the sequence of any two contiguous amino acid residuesin any one sequence motif is not repeated more than twice in thesequence motif, and wherein the content of any one amino acid type inthe full-length XTEN does not exceed 30%. In the foregoing embodimentshereinabove described in this paragraph, the XTEN sequences are“substantially non-repetitive.”

In some embodiments, the invention provides compositions comprising one,or two, or three, or four or more non-repetitive XTEN sequence(s) ofabout 36 to about 1000 amino acid residues, or cumulatively about 100 toabout 3000 amino acid residues wherein at least about 80%, or at leastabout 90%, or about 91%, or about 92%, or about 93%, or about 94%, orabout 95%, or about 96%, or about 97%, or about 98%, or about 99% toabout 100% of the sequence consists of multiple units of two or morenon-overlapping sequence motifs selected from the amino acid sequencesof Table 3, wherein the overall sequence remains substantiallynon-repetitive. In some embodiments, the XTEN comprises non-overlappingsequence motifs in which about 80%, or at least about 85%, or at leastabout 90%, or about 91%, or about 92%, or about 93%, or about 94%, orabout 95%, or about 96%, or about 97%, or about 98%, or about 99% orabout 100% of the sequence consists of multiple units of two or morenon-overlapping sequences selected from a single motif family selectedfrom Table 3, resulting in a family sequence. As used herein, “family”means that the XTEN has motifs selected only from a single motifcategory from Table 3; i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, andthat any other amino acids in the XTEN not from a family motif areselected to achieve a needed property, such as to permit incorporationof a restriction site by the encoding nucleotides, incorporation of acleavage sequence, or to achieve a better linkage to an binding proteincomponent.

TABLE 3 XTEN Sequence Motifs of 12 Amino Acids and Motif FamiliesMotif Family* MOTIF SEQUENCE SEQ ID NO: AD GESPGGSSGSES 2 ADGSEGSSGPGESS 3 AD GSSESGSSEGGP 4 AD GSGGEPSESGSS 5 AE, AM GSPAGSPTSTEE 6AE, AM, AQ GSEPATSGSETP 7 AE, AM, AQ GTSESATPESGP 8 AE, AM, AQGTSTEPSEGSAP 9 AF, AM GSTSESPSGTAP 10 AF, AM GTSTPESGSASP 11 AF, AMGTSPSGESSTAP 12 AF, AM GSTSSTAESPGP 13 AG, AM GTPGSGTASSSP 14 AG, AMGSSTPSGATGSP 15 AG, AM GSSPSASTGTGP 16 AG, AM GASPGTSSTGSP 17 AQGEPAGSPTSTSE 18 AQ GTGEPSSTPASE 19 AQ GSGPSTESAPTE 20 AQ GSETPSGPSETA 21AQ GPSETSTSEPGA 22 AQ GSPSEPTEGTSA 23 BC GSGASEPTSTEP 24 BC GSEPATSGTEPS25 BC GTSEPSTSEPGA 26 BC GTSTEPSEPGSA 27 BD GSTAGSETSTEA 28 BDGSETATSGSETA 29 BD GTSESATSESGA 30 BD GTSTEASEGSAS 31 *Denotesindividual motif sequences that, when used together in variouspermutations, results in a “family sequence”

In some embodiments of XTEN families, an XTEN sequence comprisesmultiple units of non-overlapping sequence motifs of the AD motiffamily, or an XTEN sequence comprises multiple units of non-overlappingsequence motifs of the AE motif family, or an XTEN sequence comprisesmultiple units of non-overlapping sequence motifs of the AF motiffamily, or an XTEN sequence comprises multiple units of non-overlappingsequence motifs of the AG motif family, or an XTEN sequence comprisesmultiple units of non-overlapping sequence motifs of the AM motiffamily, or an XTEN sequence comprises multiple units of non-overlappingsequence motifs of the AQ motif family, or an XTEN sequence comprisesmultiple units of non-overlapping sequence motifs of the BC family, oran XTEN sequence comprises multiple units of non-overlapping sequencemotifs of the BD family, with the resulting XTEN exhibiting the range ofhomology described above. In other embodiments, the XTEN comprisesmultiple units of motif sequences from two or more of the motif familiesof Table 3, selected to achieve desired physicochemical characteristics,including such properties as net charge, lack of secondary structure, orlack of repetitiveness that may be conferred by the amino acidcomposition of the motifs, described more fully below. In theembodiments hereinabove described in this paragraph, the motifsincorporated into the XTEN can be selected and assembled using themethods described herein to achieve an XTEN of about 36 to about 3000amino acid residues. Non-limiting examples of XTEN family sequences arepresented in Table 4.

TABLE 4 XTEN Polypeptides XTEN SEQ ID Name Amino Acid Sequence NO:AE42_1 TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS 32 AE42_2PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG 33 AE42_3SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP 34 AG42_1GAPSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGPSGP 35 AG42_2GPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASP 36 AG42_3SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA 37 AG42_4SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG 38 AE48MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS 39 AM48MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS 40 AE144GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEG 41SAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP AF144GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSG 42TAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAP AG144_PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST 43 1GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS AG144_SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP 44 2GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP AG144_GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST 45 3GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSP AG144_GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSS 46 4TGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSP AE288GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE 47SGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEG SAP AG288_ASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP 48 1GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS AG288_PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT 49 2ASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP SGATGS AG288_GSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST 50 3GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPG SGTASSSP AF504GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGA 51TGSPGSXPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSXPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP AF540GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAES 52PGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAP AD576GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSE 53GGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGSSEG GPGSEGSSGPGESSAE576 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG 54SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAP AF576GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAES 55PGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPE SGSASP AG576PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSG 56ATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS AE624MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSP 57TSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AD836GSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSS 58GSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSS AE864GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG 59SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPAF864 GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGS 60ASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP AG864GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGA 61TGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP AM875GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGS 62ASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP AE912MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSP 63TSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS AP AM923MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPS 64EGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP AM1318GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGS 65ASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEG TSTEPSEGSAPBC 864 GTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGT 66EPSGSEPATSGTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSGASEPTSTEPGTSEPSTSEPGAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSEPSTSEPGAGSGASEPTSTEPGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPG TSTEPSEPGSABD864 GSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATS 67GSETAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGSTAGSETSTEAGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETA AE948GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG 68SAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP AE1044GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTS 69TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTST AE1140GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPE 70SGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPA AE1236GSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS 71ETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEP AE1332GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGS 72ETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTST AE1428GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPE 73SGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSPA AE1524GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS 74TEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTLEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTLEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSPA AE1620GSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPE 75SGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST AE1716GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGS 76ETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSE AE1812GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGS 77ETPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEP AE1908GSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEG 78SAPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSLEEGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEP AE2004AGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGS 79ETPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTLEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTLEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTLEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTLEGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE AG948GSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTA 80SSSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP AG1044GTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSS 81TGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTP GSGTASSSPGSSTAG1140 GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSS 82TGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGT SSTGSPGSSTAG1236 GSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTA 83SSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASP GTSSTGSPGASPAG1332 GSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGTSS 84TGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGT SSTGSPGTPGAG1428 GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTA 85SSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPG SGTASSSPGASPAG1524 GSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGA 86TGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSTP SGATGSPGTPGAG1620 GSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTA 87SSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGTPGSGTASSSPGSSTPS GATGSPGSSTAG1716 GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTA 88SSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTS STGSPGTPGAG1812 GSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA 89SSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTP SGATGSPGASPAG1908 GSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSPSAST 90GTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSS PSASTGTGPGSSPAG2004A GSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGA 91TGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPS ASTGTGPGASPAE72B SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPES 92GPGSEPATSGSETPG AE72CTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST 93EEGTSTEPSEGSAPG AE108ATEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP 94SEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS AE108BGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE 95SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP AE144ASTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET 96PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS AE144BSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS 97APGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG AE180ATSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPA 98GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS APGSEPATSAE216A PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE 99SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE252AESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES 100ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE AE288ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE 101PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS ESA AE324APESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTST 102EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS AE360APESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPA 103GSPTSTLEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE396APESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA 104GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTLEGSPAGSPTSTLEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS AE432AEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE 105SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS AE468AEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSE 106SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS GSETPGTSESATAE504A EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPA 107GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS AE540ATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS 108TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP AE576ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS 109ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP GTSESA AE612AGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPA 110GSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE648APESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTST 111EPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE684AEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST 112EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS AE720ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT 113STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE AE756ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT 114STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES AE792AEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSE 115SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS AE828APESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE 116SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AG72AGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGT 117SSTGSPGTPGSGTASS AG72BGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSS 118TGSPGTPGSGTASSSP AG72CSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATG 119SPGSSTPSGATGSPGA AG108ASASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP 120GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASP AG108BPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG 121ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS AG144APGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST 122GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS AG144BPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP 123GSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASP AG180ATSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGS 124SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGT SSTGSPGTPGSAG216A TGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGAS 125PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSG AG252ATSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGS 126SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPG AG288ATSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGS 127SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS PGTPGS AG324ATSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG 128ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP AG360ATSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG 129ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG AG396AGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGT 130PGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGAS PGT AG432AGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGS 131STPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPS AG468ATSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG 132ASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPG AG504ATSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG 133ASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP AG540ATSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG 134ASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG AG576ATSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGS 135SPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPS GATGSPGASPGAG612A STGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSST 136PSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS AG648AGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPG 137SSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP AG684ATSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGS 138STPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGA TGSPGASPGAG720A TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGS 139STPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG AG756ATSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGS 140SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPG AG792ATSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGS 141SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGP GASPG AG828ATSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGS 142SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP

In other embodiments, the binding fusion protein composition comprisesone or more non-repetitive XTEN sequences of about 36 to about 3000amino acid residues, wherein at least about 80%, or at least about 90%,or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, orabout 96%, or about 97%, or about 98%, or about 99% to about 100% of thesequence consists of non-overlapping 36 amino acid sequence motifsselected from one or more of the polypeptide sequences of Tables 11-14,either as a family sequence, or where motifs are selected from two ormore families of motifs.

In those embodiments wherein the XTEN component of the fusion proteinhas less than 100% of its amino acids consisting of four to six aminoacid selected from glycine (G), alanine (A), serine (S), threonine (T),glutamate (E) and proline (P), or less than 100% of the sequenceconsisting of the sequences from any one of Tables 4 or 11-14, the otheramino acid residues are selected from any other of the 14 naturalL-amino acids, but are preferentially selected from hydrophilic aminoacids such that the XTEN sequence contains at least about 90%, or atleast about 91%, or at least about 92%, or at least about 93%, or atleast about 94%, or at least about 95%, or at least about 96%, or atleast about 97%, or at least about 98%, or at least about 99%hydrophilic amino acids. The XTEN amino acids that are not glycine (G),alanine (A), serine (S), threonine (T), glutamate (E) and proline (P)are interspersed throughout the XTEN sequence, are located within orbetween the sequence motifs, or are concentrated in one or more shortstretches of the XTEN sequence. In such cases where the XTEN componentof the binding fusion protein comprises amino acids other than glycine(G), alanine (A), serine (S), threonine (T), glutamate (E) and proline(P), it is preferred that the amino acids not be hydrophobic residuesand should not substantially confer secondary structure of the XTENcomponent. Hydrophobic residues that are less favored in construction ofXTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine,valine, and methionine. Additionally, one can design the XTEN sequencesto contain less than 5% or less than 4% or less than 3% or less than 2%or less than 1% or none of the following amino acids: cysteine (to avoiddisulfide formation and oxidation), methionine (to avoid oxidation),asparagine and glutamine (to avoid desamidation). Thus, in someembodiments, the XTEN component of the fusion protein comprising otheramino acids in addition to glycine (G), alanine (A), serine (S),threonine (T), glutamate (E) and proline (P) would have a sequence withless than 5% of the residues contributing to alpha-helices andbeta-sheets as measured by the Chou-Fasman algorithm and have at least90%, or at least about 95% or more random coil formation as measured bythe GOR algorithm.

3. Length of Sequence

In another aspect, the invention provides binding fusion proteincompositions comprising an binding protein and one or more XTENpolypeptides wherein the length of the XTEN sequences are chosen basedon the property or function to be achieved. Depending on the intendedproperty or function, the binding fusion protein compositions compriseshort or intermediate length XTEN and/or longer XTEN sequences that canserve as carriers. The subject binding fusion proteins encompass XTEN orfragments of XTEN with lengths of about 6, or about 12, or about 36, orabout 40, or about 100, or about 144, or about 288, or about 401, orabout 500, or about 600, or about 700, or about 800, or about 900, orabout 1000, or about 1500, or about 2000, or about 2500, or up to about3000 amino acid residues in length. In other cases, the XTEN sequencescan be about 6 to about 50, or about 100 to 150, about 150 to 250, about250 to 400, about 400 to about 500, about 500 to 900, about 900 to 1500,about 1500 to 2000, or about 2000 to about 3000 amino acid residues inlength. In the embodiments of the binding fusion proteins, the one ormore XTEN or fragments of XTEN sequences individually exhibit at leastabout 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity compared to a motif or an XTEN selected from anyone of Tables 4 or 11-14, or a fragment thereof with comparable length.In some embodiments, the bind fusion proteins comprise a first and atleast a second XTEN sequence, wherein the cumulative length of theresidues in the XTEN sequences is greater than about 100 to about 3000amino acid residues and the XTEN can be identical or they can bedifferent in sequence or in length. As used herein, “cumulative length”is intended to encompass the total length, in amino acid residues, whenmore than one XTEN is incorporated into the binding fusion proteins ofthe embodiments. In one embodiment of the foregoing, the first and atleast the second sequences each exhibit at least about 80% sequenceidentity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity compared to one or more XTEN sequences from Table 4, orfragments thereof.

As described more fully below, methods are disclosed in which thebinding fusion protein is designed by selecting the length of the XTENto confer a target half-life or other physicochemical property on afusion protein administered to a subject. When XTEN are used as acarrier, the invention takes advantage of the discovery that increasingthe length of the non-repetitive, unstructured polypeptides enhances theunstructured nature of the XTENs and correspondingly enhances thebiological and pharmacokinetic properties of fusion proteins comprisingthe XTEN carrier. As described more fully in the Examples, proportionalincreases in the length of the XTEN, even if created by a repeated orderof single family sequence motifs (e.g., the four AE motifs of Table 3),result in a sequence with a higher percentage of random coil formation,as determined by GOR algorithm, or reduced content of alpha-helices orbeta-sheets, as determined by Chou-Fasman algorithm, compared to shorterXTEN lengths. In addition, increasing the length of the unstructuredpolypeptide fusion partner, as described in the Examples, results in afusion protein with a disproportionate increase in terminal half-lifecompared to fusion proteins with unstructured polypeptide partners withshorter sequence lengths. In general, XTEN cumulative lengths longerthat about 400 residues incorporated into the binding fusion proteincompositions result in longer half-life compared to shorter cumulativelengths; e.g., shorter than about 280 residues.

In some embodiments, where the XTEN serve primarily as a carrier, theinvention encompasses binding fusion protein compositions comprising oneor more XTEN wherein the cumulative XTEN sequence length of the fusionprotein(s) is greater than about 100, or greater than about 200, orgreater than about 400, or greater than about 500, or greater than about600, or greater than about 800, or greater than about 900, or greaterthan about 1000 to about 3000 amino acid residues, wherein the fusionprotein exhibits enhanced pharmacokinetic properties when administeredto a subject compared to a binding protein not linked to XTEN andadministered at a comparable dose. In one embodiment of the foregoing,the one or more XTEN sequences exhibit at least about 80%, or at leastabout 90%, or at least about 91%, or at least about 92%, or at leastabout 93%, or at least about 94%, or at least about 95%, or at leastabout 96%, or at least about 97%, or at least about 98% or more identityto a sequence selected from Table 4, or fragments thereof, and theremainder of the carrier sequence(s) contain at least 90% hydrophilicamino acids and less than about 2% of the overall sequence consists ofhydrophobic or aromatic amino acids or cysteine. The enhancedpharmacokinetic properties of the binding fusion proteins in comparisonto binding proteins not linked to XTEN are described more fully, below.

4. XTEN Segments

In one aspect, the invention provides XTEN of short or intermediatelengths, wherein the choice of the XTEN confers different functions orproperties to the binding fusion proteins. In particular binding fusionprotein configuration designs, where the XTEN serve as a flexiblelinker, or are designed to interfere with clearance receptors, or wherea short or intermediate length of XTEN is used to facilitate tissuepenetration or to vary the strength of interactions of the bindingfusion protein with its target, or where it is desirable to distributethe cumulative length of XTEN in at least two segments of short orintermediate length, the invention provides binding fusion proteinscomprising one or more truncated XTEN sequences.

The XTEN of short or intermediate lengths can be an XTEN or a fragmentof an XTEN of a length of from about 6 amino acids to about 600 aminoacids, or about 12 to about 288 amino acids, or about 36 to about 144amino acids, or about 42 to about 96 amino acids in length. Non-limitingexamples of short or intermediate length XTEN contemplated for inclusionin the binding fusion proteins embodiments of the disclosure arepresented in Table 4, but can also include fragments of the motifs ofTable 3 or fragments of the sequences of Table 4 used singly or linkedin combination using the methods disclosed herein to achieve an XTEN ofa given length, including lengths encompassed by the ranges disclosedabove. In non-limiting examples, as schematically depicted in FIGS.39A-C, the AG864 sequence of 864 amino acid residues can be truncated toyield an AG144 with 144 residues, an AG288 with 288 residues, an AG576with 576 residues, or other intermediate lengths, while the AE864sequence (FIGS. 39D-E) can be truncated to yield an AE288 or AE576 orother intermediate lengths. It is specifically contemplated that such anapproach can be utilized with any of the XTEN embodiments describedherein or with any of the sequences listed in Table 4 to result in XTENof a desired length.

In another aspect, the invention provides XTEN of longer lengths whereinthe sequence is substantially non-repetitive. The incorporation oflonger length XTEN as carriers into binding fusion proteins confersenhanced properties on the fusion proteins compared to fusion partnersof shorter length XTEN, including slower rates of systemic absorption,increased bioavailability, and increased half-life after subcutaneous orintramuscular administration to a subject, and longer terminal half-lifeor area under the curve. In one embodiment, the XTEN of longer lengthshave greater than about 400, or greater than about 600, or greater thanabout 800, or greater than about 900, or greater than about 1000, orgreater than about 1100, or greater than about 1200, or greater thanabout 1300, or greater than about 1400, or greater than about 1500, orgreater than about 1600, or greater than about 1700, or greater thanabout 1800, or greater than about 1900, or greater than about 2000, upto about 3000 amino acid residues or more in length, wherein theassembled XTEN is substantially non-repetitive.

In some embodiments, the binding fusion proteins comprise at least twoXTEN segments in which the XTEN segments can be identical or they can bedifferent wherein the cumulative length of the XTEN components aregreater than about 100 to about 3000 amino acid residues and comprisesat least one sequence segment of at least about 36 to about 923, or atleast about 42 to about 875, or at least about 96 to about 576, or atleast about 100 to about 288, or at least about 132 to about 144 aminoacid residues wherein the sequence segment(s) consists of at least four,or at least five, or at least six different types of amino acids and thesum of glycine (G), alanine (A), serine (S), threonine (T), glutamate(E) and proline (P) residues in the sequence segment(s) constitutes atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 91%, or at least about 92%, or at least about 93%, or atleast about 94%, or at least about 95%, or at least about 96%, or atleast about 97%, or at least about 98%, or at least about 99% of thetotal amino acid sequence of the sequence segment and at least about90%, or at least about 91%, or at least about 92%, or at least about93%, or at least about 94%, or at least about 95%, or at least about96%, or at least about 97%, or at least about 98% of the remainder ofthe XTEN sequence(s) consist of hydrophilic amino acids and less thanabout 2% of the remainder of the XTEN sequence(s) consists ofhydrophobic or aromatic amino acids, or cysteine. In another embodiment,the invention provides an isolated binding fusion protein wherein thecumulative length of the XTEN component is greater than about 100 toabout 3000 amino acid residues and comprises at least one sequencesegment of at least about 36 to about 923, or at least about 42 to about875, or at least about 96 to about 576, or at least about 100 to about288, or at least about 132 to about 144 amino acid residues wherein thesequence segment(s) the sum of glycine (G), alanine (A), serine (S),threonine (T), glutamate (E) and proline (P) residues in the sequencesegment(s) constitutes at least about 90%, or at least about 91%, or atleast about 92%, or at least about 93%, or at least about 94%, or atleast about 95%, or at least about 96%, or at least about 97%, or atleast about 98%, or at least about 99% of the total amino acid sequenceof the sequence segment and wherein the subsequence score of a segmentor the cumulative segments is less than 10, or less than 9, or less than8, or less than 7, or less than 6, or less than 5, and at least about90%, or at least about 91%, or at least about 92%, or at least about93%, or at least about 94%, or at least about 95%, or at least about96%, or at least about 97%, or at least about 98% of the remainder ofthe XTEN sequence(s) consist of hydrophilic amino acids and less thanabout 2% of the remainder of the XTEN sequence(s) consists ofhydrophobic, aromatic or cysteine amino acids.

5. N-Terminal XTEN Expression-Enhancing Sequences

In one embodiment, the invention provides a short-length XTEN sequencedesigned to be incorporated as the N-terminal portion of the bindingfusion protein, wherein the expression of the fusion protein is enhancedin a host cell transformed with a suitable expression vector comprisingan optimized N-terminal leader sequence (that encodes the N-terminalXTEN) incorporated into the polynucleotide encoding the binding fusionprotein. It has been discovered, as described in Examples 14-17, that ahost cell transformed with such an expression vector comprising anoptimized N-terminal leader sequence (NTS) in the binding fusion proteingene results in greatly-enhanced expression of the binding fusionprotein compared to the expression of a corresponding binding fusionprotein from a polynucleotide not comprising the NTS, and can obviatethe need for incorporation of a non-XTEN leader sequence used to enhanceexpression. In one embodiment of the foregoing, the invention providesbinding fusion proteins comprising an NTS wherein the expression of thebinding fusion protein from the encoding gene in a host cell is enhancedabout 50%, or about 75%, or about 100%, or about 150%, or about 200%, orabout 400% compared to expression of a binding fusion protein notcomprising the N-terminal XTEN sequence (where the encoding gene lacksthe NTS).

In one embodiment of the foregoing, the N-terminal XTEN polypeptidecomprises a sequence that exhibits at least about 80%, more preferablyat least about 90%, more preferably at least about 91%, more preferablyat least about 92%, more preferably at least about 93%, more preferablyat least about 94%, more preferably at least about 95%, more preferablyat least about 96%, more preferably at least about 97%, more preferablyat least about 98%, more preferably at least 99%, or exhibits 100%sequence identity to the amino acid sequence of AE48 or AM48, therespective sequences as follows:

(SEQ ID NO: 143) AE48: MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTS STGS(SEQ ID NO: 144) AM48: MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSG ATGS

In another embodiment, the short-length N-terminal XTEN can be linked toan XTEN of longer length to form the N-terminal region of the bindingfusion protein, wherein the polynucleotide sequence encoding theshort-length N-terminal XTEN confers the property of enhanced expressionin the host cell, and wherein the long length of the expressed XTENcontributes to the enhanced properties of the XTEN carrier in the fusionprotein, as described above. In the foregoing, the short-length XTEN canbe linked to any of the XTEN disclosed herein (e.g., an XTEN of Table 4)and the resulting XTEN can, in turn, be linked to the N-terminal of anyof the targeting moieties disclosed herein (e.g., a targeting moietydirected to a target of Table 1) as a component of the binding fusionprotein. Alternatively, polynucleotides encoding the short-length XTEN(or its complement) can be linked to polynucleotides encoding any of theXTEN (or its complement) disclosed herein and the resulting geneencoding the N-terminal XTEN can, in turn, be linked to the 5′ end ofpolynucleotides encoding any of the targeting moieties (or to the 3′ endof its complement) disclosed herein. In preferred embodiments of theforegoing, the N-terminal XTEN polypeptide with long length can exhibitat least about 80%, or at least about 90%, or at least about 91%, or atleast about 92%, or at least about 93%, or at least about 94%, or atleast about 95%, or at least about 96%, or at least about 97%, or atleast about 98%, or at least 99%, or exhibits 100% sequence identity toan amino acid sequence selected from the group consisting of thesequences AE624, AE912, and AM923.

In any of the foregoing N-terminal XTEN embodiments described above, theN-terminal XTEN can have from about one to about six additional aminoacid residues, preferably selected from glycine, serine, threonine,glutamate, proline and alanine, to accommodate the endonucleaserestriction sites that is employed to join the nucleotides encoding theN-terminal XTEN to the gene encoding the targeting moiety of the fusionprotein. Non-limiting examples of amino acids compatible with therestrictions sites and the preferred amino acids are listed in Table 6,below. The methods for the generation of the N-terminal sequences andincorporation into the fusion proteins of the invention are describedmore fully in the Examples.

6. Net Charge

In other embodiments, the XTEN polypeptides have an unstructuredcharacteristic imparted by incorporation of amino acid residues with anet charge and containing a low proportion or no hydrophobic amino acidsin the XTEN sequence. The overall net charge and net charge density iscontrolled by modifying the content of charged amino acids in the XTENsequences, either positive or negative, with the net charge typicallyrepresented as the percentage of amino acids in the polypeptidecontributing to a charged state beyond those residues that are cancelledby a residue with an opposing charge. In some embodiments, the netcharge density of the XTEN of the compositions may be above +0.1 orbelow −0.1 charges/residue. By “net charge density” of a protein orpeptide herein is meant the net charge divided by the total number ofamino acids in the protein or propeptide. In other embodiments, the netcharge of an XTEN can be about 0%, about 1%, about 2%, about 3%, about4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 19%, or about 20% or more. In some embodiments, theXTEN sequence comprises charged residues separated by other residuessuch as serine or glycine, which leads to better expression orpurification behavior. Based on the net charge, some XTENs have anisoelectric point (pI) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, or even 6.5. In one embodiment, the XTEN will have anisoelectric point between 1.5 and 4.5 and carry a net negative chargeunder physiologic conditions.

Since most tissues and surfaces in a human or animal have a net negativecharge, in some embodiments, the XTEN sequences are designed to have anet negative charge to minimize non-specific interactions between theXTEN containing compositions and various surfaces such as blood vessels,healthy tissues, or various receptors. Not to be bound by a particulartheory, the XTEN can adopt open conformations due to electrostaticrepulsion between individual amino acids of the XTEN polypeptide thatindividually carry a net negative charge and that are distributed acrossthe sequence of the XTEN polypeptide. Such a distribution of netnegative charge in the extended sequence lengths of XTEN can lead to anunstructured conformation that, in turn, can result in an effectiveincrease in hydrodynamic radius. In preferred embodiments, the negativecharge of the subject XTEN is conferred by incorporation of glutamicacid residues. For example, where an XTEN with a negative charge isdesired, the XTEN can be selected solely from an AE family sequence,which has approximately a 17% net charge due to incorporated glutamicacid, or can include varying proportions of glutamic acid-containingmotifs of Table 3 to provide the desired degree of net charge.Non-limiting examples of AE XTEN include, but are not limited to theAE36, AE42, AE48, AE144, AE288, AE576, AE624, AE864, and AE912polypeptide sequences of Tables 4 or 12, or fragments thereof. In oneembodiment, an XTEN sequence of Tables 4 or 11-15 can be modified toinclude additional glutamic acid residues to achieve the desired netnegative charge. Accordingly, in one embodiment the invention providesXTEN in which the XTEN sequences contain about 1%, 2%, 4%, 8%, 10%, 15%,17%, 20%, 25%, or even about 30% glutamic acid. Generally, the glutamicresidues are spaced uniformly across the XTEN sequence. In some cases,the XTEN can contain about 10-80, or about 15-60, or about 20-50glutamic residues per 20 kDa of XTEN that can result in an XTEN withcharged residues that would have very similar pKa, which can increasethe charge homogeneity of the product and sharpen its isoelectric point,enhance the physicochemical properties of the resulting binding fusionprotein for, and hence, simplifying purification procedures. In oneembodiment, the invention contemplates incorporation of aspartic acidresidues into XTEN in addition to glutamic acid in order to achieve anet negative charge.

In other cases, where no net charge is desired, the XTEN can be selectedfrom, for example, AG XTEN components, such as the AG motifs of Table 3,or those AM motifs of Table 3 that have approximately no net charge.Non-limiting examples of AG XTEN include, but are not limited to AG42,AG144, AG288, AG576, and AG864 polypeptide sequences of Tables 4 or 14,or fragments thereof. In another embodiment, the XTEN can comprisevarying proportions of AE and AG motifs in order to have a net chargethat is deemed optimal for a given use or to maintain a givenphysicochemical property.

Not to be bound by a particular theory, the XTEN of the binding fusionprotein compositions with the higher net charge are expected to haveless non-specific interactions with various negatively-charged surfacessuch as blood vessels, tissues, or various receptors, which wouldfurther contribute to reduced active clearance. Conversely, it isbelieved that the XTEN of the binding fusion protein compositions with alow or no net charge would have a higher degree of interaction withsurfaces that can potentiate the activity of the associated bindingprotein, given the known contribution of phagocytic cells in theinflammatory process in the lung.

The XTEN of the compositions of the present invention generally have noor a low content of positively charged amino acids. In some embodiments,the XTEN may have less than about 10% amino acid residues with apositive charge, or less than about 7%, or less than about 5%, or lessthan about 2%, or less than about 1% amino acid residues with a positivecharge. However, the invention contemplates constructs where a limitednumber of amino acids with a positive charge, such as lysine, areincorporated into XTEN to permit conjugation between the epsilon amineof the lysine and a reactive group on a peptide, a linker bridge, or areactive group on a drug or small molecule to be conjugated to the XTENbackbone. In one embodiment of the foregoing, the XTEN has between about1 to about 100 lysine residues, or about 1 to about 70 lysine residues,or about 1 to about 50 lysine residues, or about 1 to about 30 lysineresidues, or about 1 to about 20 lysine residues, or about 1 to about 10lysine residues, or about 1 to about 5 lysine residues, or alternativelyonly a single lysine residue. Using the foregoing lysine-containingXTEN, fusion proteins are constructed that comprises XTEN, a bindingprotein, plus a chemotherapeutic agent useful in the treatment ofdiseases or disorders, wherein the maximum number of molecules of theagent incorporated into the XTEN component is determined by the numbersof lysines or other amino acids with reactive side chains (e.g.,cysteine) incorporated into the XTEN.

As hydrophobic amino acids impart structure to a polypeptide, theinvention provides that the content of hydrophobic amino acids in theXTEN will typically be less than 5%, or less than 2%, or less than 1%hydrophobic amino acid content. In one embodiment, the amino acidcontent of methionine and tryptophan in the XTEN component of a bindingfusion protein is typically less than 5%, or less than 2%, and mostpreferably less than 1%. In another embodiment, the XTEN will have asequence that has less than 10% amino acid residues with a positivecharge, or less than about 7%, or less that about 5%, or less than about2% amino acid residues with a positive charge, the sum of methionine andtryptophan residues will be less than 2%, and the sum of asparagine andglutamine residues will be less than 10% of the total XTEN sequence.

7. Low Immunogenicity

In another aspect, the invention provides compositions in which the XTENsequences have a low degree of immunogenicity or are substantiallynon-immunogenic. Several factors can contribute to the lowimmunogenicity of XTEN, e.g., the non-repetitive sequence, theunstructured conformation, the high degree of solubility, the low degreeor lack of self-aggregation, the low degree or lack of proteolytic siteswithin the sequence, and the low degree or lack of epitopes in the XTENsequence.

Conformational epitopes are formed by regions of the protein surfacethat are composed of multiple discontinuous amino acid sequences of theprotein antigen. The precise folding of the protein brings thesesequences into a well-defined, stable spatial configurations, orepitopes, that can be recognized as “foreign” by the host humoral immunesystem, resulting in the production of antibodies to the protein ortriggering a cell-mediated immune response. In the latter case, theimmune response to a protein in an individual is heavily influenced byT-cell epitope recognition that is a function of the peptide bindingspecificity of that individual's HLA-DR allotype. Engagement of a MHCClass II peptide complex by a cognate T-cell receptor on the surface ofthe T-cell, together with the cross-binding of certain otherco-receptors such as the CD4 molecule, can induce an activated statewithin the T-cell. Activation leads to the release of cytokines furtheractivating other lymphocytes such as B cells to produce antibodies oractivating T killer cells as a full cellular immune response.

The ability of a peptide to bind a given MHC Class II molecule forpresentation on the surface of an APC (antigen presenting cell) isdependent on a number of factors; most notably its primary sequence. Inone embodiment, a lower degree of immunogenicity may be achieved bydesigning XTEN sequences that resist antigen processing in antigenpresenting cells, and/or choosing sequences that do not bind MHCreceptors well. The invention provides binding fusion proteins withsubstantially non-repetitive XTEN polypeptides designed to reducebinding with MHC II receptors, as well as avoiding formation of epitopesfor T-cell receptor or antibody binding, resulting in a low degree ofimmunogenicity. Avoidance of immunogenicity is, in part, a direct resultof the conformational flexibility of XTEN sequences; i.e., the lack ofsecondary structure due to the selection and order of amino acidresidues. For example, of particular interest are sequences having a lowtendency to adapt compactly folded conformations in aqueous solution orunder physiologic conditions that could result in conformationalepitopes. The administration of fusion proteins comprising XTEN, usingconventional therapeutic practices and dosing, would generally notresult in the formation of neutralizing antibodies to the XTEN sequence,and may also reduce the immunogenicity of the targeting moiety fusionpartner in the binding fusion protein compositions.

In one embodiment, the XTEN sequences utilized in the subject fusionproteins can be substantially free of epitopes recognized by human Tcells. The elimination of such epitopes for the purpose of generatingless immunogenic proteins has been disclosed previously; see for exampleWO 98/52976, WO 02/079232, and WO 00/3317 which are incorporated byreference herein. Assays for human T cell epitopes have been described(Stickler, M., et al. (2003) J Immunol Methods, 281: 95-108). Ofparticular interest are peptide sequences that can be oligomerizedwithout generating T cell epitopes or non-human sequences. This can beachieved by testing direct repeats of these sequences for the presenceof T-cell epitopes and for the occurrence of 6 to 15-mer and, inparticular, 9-mer sequences that are not human, and then altering thedesign of the XTEN sequence to eliminate or disrupt the epitopesequence. In one embodiment, the XTEN sequences are substantiallynon-immunogenic by the restriction of the numbers of epitopes of theXTEN predicted to bind MHC receptors. With a reduction in the numbers ofepitopes capable of binding to MHC receptors, there is a concomitantreduction in the potential for T cell activation as well as T cellhelper function, reduced B cell activation or upregulation and reducedantibody production. The low degree of predicted T-cell epitopes can bedetermined by epitope prediction algorithms such as, e.g., TEPITOPE(Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555-61), as shown inExample 59. The TEPITOPE score of a given peptide frame within a proteinis the log of the K_(d) (dissociation constant, affinity, off-rate) ofthe binding of that peptide frame to multiple of the most common humanMHC alleles, as disclosed in Sturniolo, T. et al. (1999) NatureBiotechnology 17:555). The score ranges over at least 20 logs, fromabout 10 to about −10 (corresponding to binding constraints of 10e¹⁰K_(d) to 10e⁻¹⁰ K_(d)), and can be reduced by avoiding hydrophobic aminoacids that can serve as anchor residues during peptide display on MHC,such as M, I, L, V, F. In some embodiments, an XTEN componentincorporated into a binding fusion protein does not have a predictedT-cell epitope at a TEPITOPE threshold score of about −5, or −6, or −7,or −8, or −9, or at a TEPITOPE score of −10. As used herein, a score of“−9” would be a more stringent TEPITOPE threshold than a score of −5.

In another embodiment, the inventive XTEN sequences, including thoseincorporated into the subject binding fusion proteins, can be renderedsubstantially non-immunogenic by the restriction of known proteolyticsites from the sequence of the XTEN, reducing the processing of XTENinto small peptides that can bind to MHC II receptors. In anotherembodiment, the XTEN sequence can be rendered substantiallynon-immunogenic by the use a sequence that is substantially devoid ofsecondary structure, conferring resistance to many proteases due to thehigh entropy of the structure. Accordingly, the reduced TEPITOPE scoreand elimination of known proteolytic sites from the XTEN may render theXTEN compositions, including the XTEN of the binding fusion proteincompositions, substantially unable to be bound by mammalian receptors,including those of the immune system. In one embodiment, an XTEN of abinding fusion protein can have >100 nM K_(d) binding to a mammalianreceptor, or greater than 500 nM K_(d), or greater than 1 μM K_(d)towards a mammalian cell surface or circulating polypeptide receptor.

Additionally, the non-repetitive sequence and corresponding lack ofepitopes of XTEN can limit the ability of B cells to bind to or beactivated by XTEN. A repetitive sequence is recognized and can formmultivalent contacts with even a few B cells and, as a consequence ofthe cross-linking of multiple T-cell independent receptors, canstimulate B cell proliferation and antibody production. In contrast,while a XTEN can make contacts with many different B cells over itsextended sequence, each individual B cell may only make one or a smallnumber of contacts with an individual XTEN due to the lack ofrepetitiveness of the sequence. As a result, XTENs typically may have amuch lower tendency to stimulate proliferation of B cells and thus animmune response. In one embodiment, the binding fusion protein may havereduced immunogenicity as compared to the corresponding targeting moietythat is not fused. In one embodiment, the administration of up to threeparenteral doses of a binding fusion protein to a mammal may result indetectable anti-binding fusion protein IgG at a serum dilution of 1:100but not at a dilution of 1:1000. In another embodiment, theadministration of up to three parenteral doses of a binding fusionprotein to a mammal may result in detectable anti-targeting moiety IgGat a serum dilution of 1:100 but not at a dilution of 1:1000. In anotherembodiment, the administration of up to three parenteral doses of abinding fusion protein to a mammal may result in detectable anti-XTENIgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In theforegoing embodiments, the mammal can be a mouse, a rat, a rabbit, or acynomolgus monkey.

An additional feature of XTENs with non-repetitive sequences relative tosequences with a high degree of repetitiveness can be thatnon-repetitive XTENs form weaker contacts with antibodies. Antibodiesare multivalent molecules. For instance, IgGs have two identical bindingsites and IgMs contain 10 identical binding sites. Thus antibodiesagainst repetitive sequences can form multivalent contacts with suchrepetitive sequences with high avidity, which can affect the potencyand/or elimination of such repetitive sequences. In contrast, antibodiesagainst non-repetitive XTENs may yield monovalent interactions,resulting in less likelihood of immune clearance such that the bindingfusion protein compositions can remain in circulation for an increasedperiod of time.

8. Increased Hydrodynamic Radius

In another aspect, the present invention provides XTEN in which the XTENpolypeptides can have a high hydrodynamic radius that confers acorresponding increased apparent molecular weight factor to fusionprotein incorporating the XTEN. As detailed in Example 40, the linkingof XTEN to targeting moiety sequences can result in binding fusionprotein compositions that can have increased hydrodynamic radii,increased apparent molecular weight factor, and increased apparentmolecular weight factor compared to a targeting moiety not linked to anXTEN. For example, in therapeutic applications in which prolongedhalf-life is desired, compositions in which a XTEN with a highhydrodynamic radius is incorporated into a fusion protein comprising oneor more targeting moieties can effectively enlarge the hydrodynamicradius of the composition beyond the glomerular pore size ofapproximately 3-5 nm (corresponding to an apparent molecular weight ofabout 70 kDA) (Caliceti. 2003. Pharmacokinetic and biodistributionproperties of poly(ethylene glycol)-protein conjugates. Adv Drug DelivRev 55:1261-1277), resulting in reduced renal clearance of circulatingproteins. The hydrodynamic radius of a protein is determined by itsmolecular weight as well as by its structure, including shape andcompactness. Not to be bound by a particular theory, the XTEN can adoptopen conformations due to electrostatic repulsion between individualcharges of the peptide or the inherent flexibility imparted by theparticular amino acids in the sequence that lack potential to confersecondary structure. The open, extended and unstructured conformation ofthe XTEN polypeptide can have a greater proportional hydrodynamic radiuscompared to polypeptides of a comparable sequence length and/ormolecular weight that have secondary and/or tertiary structure, such astypical globular proteins. Methods for determining the hydrodynamicradius are well known in the art, such as by the use of size exclusionchromatography (SEC), as described in U.S. Pat. Nos. 6,406,632 and7,294,513. As the results of Example 40 demonstrate, the addition ofincreasing lengths of XTEN to a payload polypeptide results inproportional increases in the parameters of hydrodynamic radius,apparent molecular weight factor, and apparent molecular weight factor,permitting the tailoring of binding fusion proteins to desiredcharacteristic cut-off apparent molecular weight factors or hydrodynamicradii. Accordingly, in certain embodiments, the binding fusion proteincan be configured with an XTEN such that the fusion protein can have ahydrodynamic radius of at least about 5 nm, or at least about 8 nm, orat least about 10 nm, or 12 nm, or at least about 15 nm. In theforegoing embodiments, the large hydrodynamic radius conferred by theXTEN in a binding fusion protein can lead to reduced renal clearance ofthe resulting fusion protein, leading to a corresponding increase interminal half-life, an increase in mean residence time, and/or adecrease in renal clearance rate.

In another embodiment, an XTEN of a chosen length and sequence can beselectively incorporated into a binding fusion protein to create afusion protein that will have, under physiologic conditions, an apparentmolecular weight of at least about 150 kDa, or at least about 300 kDa,or at least about 400 kDa, or at least about 500 kDA, or at least about600 kDa, or at least about 700 kDA, or at least about 800 kDa, or atleast about 900 kDa, or at least about 1000 kDa, or at least about 1200kDa, or at least about 1500 kDa, or at least about 1800 kDa, or at leastabout 2000 kDa, or at least about 2300 kDa or more. In anotherembodiment, an XTEN of a chosen length and sequence can be selectivelylinked to a targeting moiety to result in a binding fusion protein thathas, under physiologic conditions, an apparent molecular weight factorof at least three, alternatively of at least four, alternatively of atleast five, alternatively of at least six, alternatively of at leasteight, alternatively of at least 10, alternatively of at least 15, or anapparent molecular weight factor of at least 20 or greater. In anotherembodiment, the binding fusion protein has, under physiologicconditions, an apparent molecular weight factor that is about 4 to about20, or is about 6 to about 15, or is about 8 to about 12, or is about 9to about 10 relative to the actual molecular weight of the fusionprotein.

(c) Targeting Moieties

In another aspect of the invention, targeting moieties are disclosedthat can be linked to one or more XTEN, resulting in monomeric bindingfusion protein compositions. “Targeting moieties”, as used herein,refers to polypeptides that have specific binding affinity for a targetligand such as cytokines, chemokines, cytokine receptors, chemokinesreceptors, hormones, cell-surface receptors or antigens orglycoproteins, oligonucleotides, enzymatic substrates, antigenicdeterminants, or other binding sites that may be present in thecirculation, or on the surface or in the cytoplasm of a target cell.Non-limiting, exemplary targets to which the targeting moieties of thesubject compositions are directed are disclosed above; e.g., targetsselected from Table 1 and Table 2. The invention provides multiplecategories of targeting moieties that can be linked to one or more XTENin various configurations, resulting in the inventive binding fusionprotein compositions. As described more fully below, the targetingmoieties can be derived from or based on sequences of antibodies,antibody fragments, receptors, immunoglobulin-like binding domains, orcan be completely synthetic. The binding fusion proteins can compriseone or more functional antigen binding sites, the latter making thebinding fusion protein “multivalent.” An “antigen binding site” of abinding fusion protein is one that is capable of binding a targetantigen with at least a portion of the binding affinity of the parentalantibody or receptor from which the antigen binding site is derived. Theantigen binding site may itself be composed of more than one bindingdomain, linked together in the binding fusion proteins. “Binding domain”means a polypeptide sequence capable of attaching to an antigen orligand but that may require additional binding domains to actually bindand/or sequester the antigen or ligand. A CDR from an antibody is anexample of a binding domain. “Antibody” is used throughout thespecification as a prototypical example of a targeting moiety but is notintended to be limiting.

Methods to measure binding affinity and/or other biologic activity ofthe binding fusion protein compositions of the invention can be thosedisclosed herein or methods generally known in the art. In addition, thephysicochemical properties of the binding fusion protein may be measuredto ascertain the degree of solubility, structure and retention ofstability. Assays are conducted that allow determination of bindingcharacteristics of the targeting moieties towards a ligand, includingbinding dissociation constant (K_(d), K_(on) and K_(off)), the half-lifeof dissociation of the ligand-receptor complex, as well as the activityof the binding fusion protein to inhibit the biologic activity of thesequestered ligand compared to free ligand (IC₅₀ values). The term“K_(d)”, as used herein, is intended to refer to the dissociationconstant of a particular antibody-antigen interaction as is known in theart, and would apply as a parameter of the binding affinity of atargeting moiety to its cognate ligand for the subject compositions. Theterm “K_(on)”, as used herein, is intended to refer to the on rateconstant for association of an antibody to the antigen to form theantibody/antigen complex as is known in the art. The term “K_(off)”, asused herein, is intended to refer to the off rate constant fordissociation of an antibody from the antibody/antigen complex as isknown in the art. The term “IC₅₀” refers to the concentration needed toinhibit half of the maximum biological response of the ligand agonist,and is generally determined by competition binding assays.

Techniques such as flow cytometry or surface plasmon resonance can beused to detect binding events. The assays may comprise soluble antigensor receptor molecules, or may determine the binding to cell-expressedreceptors. Such assays may include cell-based assays, including assaysfor proliferation, cell death, apoptosis and cell migration. The bindingaffinity of the subject compositions for the target ligands can beassayed using binding or competitive binding assays, such as Biacoreassays with chip-bound receptors or binding proteins or ELISA assays, asdescribed in U.S. Pat. No. 5,534,617, assays described in the Examplesherein, radio-receptor assays, or other assays known in the art. Thebinding affinity constant can then be determined using standard methods,such as Scatchard analysis, as described by van Zoelen, et al., TrendsPharmacol Sciences (1998) 19)12):487, or other methods known in the art.In addition, libraries of sequence variants of targeting moieties can becompared to the corresponding native or parental antibodies using acompetitive ELISA binding assay to determine whether they have the samebinding specificity and affinity as the parental antibody, or somefraction thereof such that they are suitable for inclusion in thebinding fusion proteins. The results of such assays can be used in aniterative process of sequence modification of the targeting moietiesfollowed by binding and physicochemical characterization assays to guidethe process by which specific constructs with the desired properties areselected.

In one embodiment, the invention provides isolated binding fusionproteins that competitively inhibit binding of an antibody to a targetligand, as determined by any method known in the art for determiningcompetitive binding, such as the immunoassays described herein. Theantibody can include the parental antibody from with the targetingmoiety was derived or a positive control known to bind the targetepitope or ligand. In preferred embodiments, the binding fusion proteincompetitively inhibits binding of the positive control to the ligand byat least 90%, at least 80%, at least 70%, at least 60%, or at least 50%in a competitive binding assay against the positive control.

The invention provides isolated binding fusion proteins in which thebinding affinity of the one or more targeting moieties for targetligands can be at least about 1%, or at least about 10%, or at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90%, or at least about 95%, or at leastabout 99%, or at least about 99.9% or more of the affinity of a parentalantibody not bound to XTEN for the target receptor or ligand. In oneembodiment, the K_(d) between the one or more targeting moieties of thesubject binding fusion protein and a target ligand is less than about10⁻⁴ M, alternatively less than about 10⁻⁵M, alternatively less thanabout 10⁻⁶M, alternatively less than about 10⁻⁷M, alternatively lessthan about 10⁻⁸M, alternatively less than about 10⁻⁹M, or less thanabout 10⁻¹⁰ M, or less than about 10⁻¹¹M, or less than about 10⁻¹²M. Inthe foregoing embodiment, the binding affinity of the binding fusionprotein towards the target would be characterized as “specific.” Theinvention contemplates binding fusion proteins comprising two or moretargeting moieties in which the binding affinities for the respectivetargeting moieties may independently be between the ranges of values ofthe foregoing. In any of the foregoing embodiments of the paragraph, theone or more targeting moieties of the subject binding fusion proteinsspecifically bind to a target of Table 1 or Table 2.

Binding fusion proteins of the present invention may also be describedor specified in terms of the cross-reactivity of the targeting moiety.In one embodiment, the invention provides binding fusion proteins thatdo not bind any other analog, ortholog, or homolog of a target disclosedherein. In one embodiment, binding fusion proteins can bind polypeptideswith at least 95%, at least 90%, at least 85%, at least 80%, at least75%, at least 70%, at least 65%, at least 60%, at least 55%, and atleast 50% sequence identity (as calculated using methods known in theart and described herein) to a polypeptide target described herein.

The binding fusion proteins of the present invention may act as agonistsor antagonists. For example, the present invention includes bindingfusion proteins comprising targeting moieties that disruptreceptor/ligand interactions either partially or fully. The inventionfeatures both receptor-specific BFP and ligand-specific BFP. Theinvention also features receptor-specific binding fusion proteins thatdo not prevent ligand binding but prevent receptor activation. Receptoractivation, such as cell signaling, may be determined by techniquesdescribed herein or otherwise known in the art. For example, receptoractivation can be determined by detecting the phosphorylation (e.g.,tyrosine or serine/threonine) of the receptor or its substrate byimmunoprecipitation followed by standard Western blot analysistechniques. In specific embodiments, binding fusion proteins areprovided that can bind to and inhibit ligand or receptor activity by atleast 90%, at least 80%, at least 70%, at least 60%, or at least 50% ofthe activity compared to the activity in the absence of the bindingfusion protein.

The invention provides receptor-specific binding fusion proteins thatboth prevent ligand binding and receptor activation as well as bindingfusion proteins that recognize the receptor-ligand complex, yet,preferably, do not specifically recognize the unbound receptor or theunbound ligand. In one embodiment, the invention provides neutralizingbinding fusion proteins that bind the ligand, thereby forming aneutralizing complex that prevents binding of the ligand to thereceptor, or, in other cases, can bind the ligand but do not prevent theligand from binding the receptor, yet nevertheless result in reducedreceptor activation in comparison to non-complexed ligand. Furtherincluded in the invention are binding fusion proteins that activate thereceptor. These BFP may act as receptor agonists, i.e., potentiate oractivate either all or a subset of the biological activities of theligand-mediated receptor activation.

In another embodiment, the invention provides isolated binding fusionproteins in which the fusion protein is designed to bind with highaffinity to a target receptor, thereby resulting in antagonisticactivity for the native ligand. In such cases, the BFPs can haveaffinity but no efficacy for their cognate receptors such that theirbinding will disrupt the interaction and inhibit the function of anagonist or inverse agonist at the receptors. Typically, suchantagonistic activity will be of a competitive type, and a K, can bedetermined. A non-limiting example of an antagonist BFP is a fusionprotein comprising a targeting moiety configured to bind to an IL-1receptor (IL-1R) such that the bound composition substantiallyinterferes with the binding of IL-1α and/or IL-1β to IL-1 receptor. Incertain cases, the interference by an antagonist binding fusion protein(such as, but not limited to an anti-IL-1R binding fusion protein) withthe binding of the native ligand to its cognate receptor can be at leastabout 1%, or about 10%, or about 20%, or about 30%, or about 40%, orabout 50%, or about 60%, or about 70%, or about 80%, or about 90%, orabout 95%, or about 99% or more. In other embodiments, the inventionprovides isolated binding fusion proteins (such as, but not limited toanti-IL-1R binding fusion protein) wherein the binding of the isolatedfusion protein to a cellular receptor elicits less than 20%, or lessthan 10%, or less than 5% activation of the signaling pathways of thecell with bound binding fusion protein antagonist in comparison to thoseevoked by the native ligand.

In one embodiment, the invention provides isolated binding fusionproteins comprising targeting moieties directed to one or more targetcytokines, cytokine-related proteins, cytokine receptors, chemokines,chemokines receptors, cell surface receptors, hormones or similarcirculating proteins or peptides, oligonucleotides, or enzymaticsubstrates. In one embodiment, the one or more targeting moieties canhave specific binding affinity to targets selected from, but not limitedto the targets of Table 1. In another embodiment, the one or moretargeting moieties can have specific binding affinity to targetsselected from, but not limited to the tumor associated antigen targetsof Table 2.

The present invention provides a variety of binding fusion proteinconfigurations in which the variations are based on inclusion of thetype and relative position or number of binding domains, as well as theinclusion of linkers of pre-determined length and one or more XTENsequences. By design, the resulting binding fusion protein compositionscan be monomeric or multivalent, they can bind a single ligand orantigen, or be multimeric as to the number of binding units encompassedin the fusion protein.

In one embodiment, the invention provides binding fusion proteinscomprising targeting moieties capable of binding to a single ligand. Inanother embodiment, the binding fusion proteins of the invention aremultivalent and the targeting moieties specifically bind at least twodifferent target antigens or ligands (“bifunctional” or “bispecific”),or different epitopes on the same ligand. The multivalent binding fusionproteins can be designed to be bifunctional in that they can incorporateheterologous binding domains from different “parental” antibodies andbind two different ligands or antigens in order to better effect adesired pharmacological response; e.g., dimerization of receptors on atarget cell surface leading to cell signaling or, alternatively, celldeath, or modulating a biological function of one or more targets.Multimeric binding fusion protein leading to cell death, whether bytriggering apoptosis or necrosis, are expected to have utility in,particularly, the treatment of oncological disease. Non-limitingexamples of pairs of targets contemplated as suitable for multivalent,bifunctional binding fusion proteins include: IGF1 and IGF2; IGF1/2 andErb2B; VEGFR and EGFR; CD20 and CD3, CD138 and CD20, CD38 and CD20, CD38& CD138, CD40 and CD20, CD138 and CD40, CD38 and CD40.

In one embodiment, the binding fusion proteins of the inventionspecifically bind at least two cytokines, lymphokines, monokines, and/orpolypeptide hormones. Non-limiting examples of pairs of targets to whichbifunctional binding fusion proteins can bind are selected from, but notlimited to IL-1α and IL-1β; IL-12 and IL-18, TNFα and IL-23, TNFα andIL-13; TNF and IL-18; TNF and IL-12; TNF and IL-1beta; TNF and MIF; TNFand IL-17; and TNF and IL-15; TNF and VEGF; VEGFR and EGFR; IL-13 andIL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC;IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHR agonist;IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAM8; andTNFα and PGE4, IL-13 and PED2, TNF and PEG2.

Examples of other pairs of targets suitable for multivalent bifunctionalbinding fusion proteins, include but are not limited to CD19 and CD20;CD-8 and IL-6; PDL-1 and CTLA-4; CTLA-4 and BTNO2; CSPGs and RGM A;IL-12 and IL-18; IL-12 and TWEAK; IL-13 and ADAM8; IL-13 and CL25; IL-13and IL-1beta; IL-13 and IL-25; IL-13 and IL-4; IL-13 and IL-5; IL-13 andIL-9; IL-13 and LHR agonist; IL-13 and MDC; IL-13 and MIF; IL-13 andPED2; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and TARC; IL-13 andTGF-β; IL-1α and IL-1β; MAG and RGM A; NgR and RGM A; NogoA and RGM A;OMGp and RGM A; RGM A and RGM B; Te38 and TNFα; TNFα and IL-12; TNFα andIL-12p40; TNFα and IL-13; TNFα and IL-15; TNFα and IL-17; TNFα andIL-18; TNFα and IL-1beta; TNFα and IL-23; TNFα and MIF; TNFα and PEG2;TNFα and PGE4; TNFα and VEGF; TNFα and RANK ligand; TNFα and Blys; TNFαand GP130; TNFα and CD-22; and TNFα and CTLA-4.

In another embodiment the binding fusion proteins of the inventionspecifically bind to pairs of targets selected from, but not limited toCD138 and CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD38 &CD138 CD38 and CD20; CD38 and CD40; CD40 and CD20; CD-8 and IL-6; CSPGsand RGM A; CTLA4 and BTNO2; IGF1 and IGF2; IGF1/2 and Erb2B; IL-12 andTWEAK; IL-13 and IL-1β; MAG and RGM A; NgR and RGM A; NogoA and RGM A;OMGp and RGM A; PDL-1 and CTLA4; RGM A and RGM B; Te38 and TNFα; TNFαand Blys; TNFα and CD-22; TNFα and CTLA-4; TNFα and GP130; TNFα andIL-12p40; and TNFα and RANK ligand.

The targeting moieties of the binding fusion proteins can be derivedfrom one or more fragments of various monoclonal antibodies known in theart. Non-limiting examples of such monoclonal antibodies include, butare not limited to anti-TNF antibody (U.S. Pat. No. 6,258,562),anti-IL-12 and or anti-IL-12p40 antibody (U.S. Pat. No. 6,914,128);anti-IL-18 antibody (US 2005/0147610 A1), anti-RANKL (U.S. Pat. No.7,411,050), anti-05, anti-CBL, anti-CD147, anti-gp120, anti-VLA4,anti-CD11a, anti-CD18, anti-VEGF, anti-CD40L, anti-Id, anti-ICAM-1,anti-CXCL13, anti-CD2, anti-EGFR, anti-TGF-beta 2, anti-E-selectin,anti-Fact VII, anti-Her2/neu, anti-Fgp, anti-CD11/18, anti-CD14,anti-ICAM-3, anti-CD80, anti-CD4, anti-CD3, anti-CD23,anti-beta2-integrin, anti-alpha4beta7, anti-CD52, anti-HLA DR,anti-CD22, anti-CD20, anti-MIF, anti-CD64 (FcR), anti-TCR alpha beta,anti-CD2, anti-Hep B, anti-CA 125, anti-EpCAM, anti-gp120, anti-CMV,anti-anti-IgE, anti-CD25, anti-CD33, anti-HLA, anti-VNRintegrin,anti-IL-1alpha, anti-IL-1beta, anti-IL-1 receptor, anti-IL-2 receptor,anti-IL-4, anti-IL4 receptor, anti-IL5, anti-IL-5 receptor, anti-IL-6,anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13 receptor, anti-IL-17, andanti-IL-23 (see Presta L G. 2005 Selection, design, and engineering oftherapeutic antibodies J Allergy Clin Immunol. 116:731-6 and Clark, M.,“Antibodies for Therapeutic Applications,” Department of Pathology,Cambridge University, UK, 15 Oct. 2000, published online at M. Clark'shome page at the website for the Department of Pathology, CambridgeUniversity).

In some embodiments, the targeting moieties are derived from one or morefragments of therapeutic monoclonal antibodies approved for use inhumans or antibodies that have demonstrated efficacy in clinical trialsor established preclinical models of diseases, disorders or conditions.Such therapeutic antibodies include, but are not limited to, rituximab,IDEC/Genentech/Roche (see for example U.S. Pat. No. 5,736,137), achimeric anti-CD20 antibody used in the treatment of many lymphomas,leukemias, and some autoimmune disorders; ofatumumab, an anti-CD20antibody approved for use for chronic lymphocytic leukemia, and underdevelopment for follicular non-Hodgkin's lymphoma, diffuse large B celllymphoma, rheumatoid arthritis and relapsing remitting multiplesclerosis, being developed by GlaxoSmithKline; lucatumumab (HCD122), ananti-CD40 antibody developed by Novartis for Non-Hodgkin's or Hodgkin'sLymphoma (see, for example, U.S. Pat. No. 6,899,879), AME-133, anantibody developed by Applied Molecular Evolution which binds to cellsexpressing CD20 to treat non-Hodgkin's lymphoma, veltuzumab (hA20), anantibody developed by Immunomedics, Inc. which binds to cells expressingCD20 to treat immune thrombocytopenic purpura, HumaLYM developed byIntracel for the treatment of low-grade B-cell lymphoma, andocrelizumab, developed by Genentech which is an anti-CD20 monoclonalantibody for treatment of rheumatoid arthritis (see for example U.S.Patent Application 20090155257), trastuzumab (see for example U.S. Pat.No. 5,677,171), a humanized anti-Her2/neu antibody approved to treatbreast cancer developed by Genentech; pertuzumab, an anti-Her2dimerization inhibitor antibody developed by Genentech in treatment ofin prostate, breast, and ovarian cancers; (see for example U.S. Pat. No.4,753,894); cetuximab, an anti-EGRF antibody used to treat epidermalgrowth factor receptor (EGFR)-expressing, KRAS wild-type metastaticcolorectal cancer and head and neck cancer, developed by Imclone and BMS(see U.S. Pat. No. 4,943,533; PCT WO 96/40210); panitumumab, a fullyhuman monoclonal antibody specific to the epidermal growth factorreceptor (also known as EGF receptor, EGFR, ErbB-1 and Her1, currentlymarketed by Amgen for treatment of metastatic colorectal cancer (seeU.S. Pat. No. 6,235,883); zalutumumab, a fully human IgG1 monoclonalantibody developed by Genmab that is directed towards the epidermalgrowth factor receptor (EGFR) for the treatment of squamous cellcarcinoma of the head and neck (see for example U.S. Pat. No.7,247,301); nimotuzumab, a chimeric antibody to EGFR developed byBiocon, YM Biosciences, Cuba, and Oncosciences, Europe) in the treatmentof squamous cell carcinomas of the head and neck, nasopharyngeal cancerand glioma (see for example U.S. Pat. Nos. 5,891,996; 6,506,883);alemtuzumab, a humanized monoclonal antibody to CD52 marketed by BayerSchering Pharma for the treatment of chronic lymphocytic leukemia (CLL),cutaneous T-cell lymphoma (CTCL) and T-cell lymphoma; muromonab-CD3, ananti-CD3 antibody developed by Ortho Biotech/Johnson & Johnson used asan immunosuppressant biologic given to reduce acute rejection inpatients with organ transplants; ibritumomab tiuxetan, an anti-CD20monoclonal antibody developed by IDEC/Schering AG as treatment for someforms of B cell non-Hodgkin's lymphoma; gemtuzumab ozogamicin, ananti-CD33 (p67 protein) antibody linked to a cytotoxic chelatortiuxetan, to which a radioactive isotope is attached, developed byCelltech/Wyeth used to treat acute myelogenous leukemia; alefacept, ananti-LFA-3 Fc fusion developed by Biogen that is used to controlinflammation in moderate to severe psoriasis with plaque formation;abciximab, made from the Fab fragments of an antibody to the IIb/IIIareceptor on the platelet membrane developed by Centocor/Lilly as aplatelet aggregation inhibitor mainly used during and after coronaryartery procedures; basiliximab, a chimeric mouse-human monoclonalantibody to the α chain (CD25) of the IL-2 receptor of T cells,developed by Novartis, used to prevent rejection in organtransplantation; palivizumab, developed by Medimmune; infliximab(REMICADE), an anti-TNFalpha antibody developed by Centocor/Johnson andJohnson, adalimumab (HUMIRA), an anti-TNFalpha antibody developed byAbbott, HUMICADE, an anti-TNFalpha antibody developed by Celltech,etanercept (ENBREL), an anti-TNFalpha Fc fusion developed byImmunex/Amgen, ABX-CBL, an anti-CD147 antibody developed by Abgenix,ABX-IL8, an anti-IL8 antibody developed by Abgenix, ABX-MA1, ananti-MUC18 antibody developed by Abgenix, Pemtumomab (R1549,90Y-muHMFG1), an anti-MUC1 in development by Antisoma, Therex (R1550),an anti-MUC1 antibody developed by Antisoma, AngioMab (AS1405),developed by Antisoma, HuBC-1, developed by Antisoma, Thioplatin(AS1407) developed by Antisoma, ANTEGREN (natalizumab), ananti-alpha-4-beta-1 (VLA4) and alpha-4-beta-7 antibody developed byBiogen, VLA-1 mAb, an anti-VLA-1 integrin antibody developed by Biogen,LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody developed byBiogen, CAT-152, an anti-TGF-β2 antibody developed by Cambridge AntibodyTechnology, J695, an anti-IL-12 antibody developed by Cambridge AntibodyTechnology and Abbott, CAT-192, an anti-TGFβ1 antibody developed byCambridge Antibody Technology and Genzyme, CAT-213, an anti-Eotaxin1antibody developed by Cambridge Antibody Technology, LYMPHOSTAT-B, ananti-Blys antibody developed by Cambridge Antibody Technology and HumanGenome Sciences Inc., TRAIL-R1mAb, an anti-TRAIL-R1 antibody developedby Cambridge Antibody Technology and Human Genome Sciences, Inc.,bevacizumab (AVASTIN, rhuMAb-VEGF), an anti-VEGF antibody developed byGenentech, HERCEPTIN, an anti-HER receptor family antibody developed byGenentech, Anti-Tissue Factor (ATF), an anti-Tissue Factor antibodydeveloped by Genentech, XOLAIR (Omalizumab), an anti-IgE antibodydeveloped by Genentech, MLN-02 Antibody (formerly LDP-02), developed byGenentech and Millennium Pharmaceuticals, HUMAX CD4®, an anti-CD4antibody developed by Genmab, tocilizuma, and anti-IL6R antibodydeveloped by Chugai, HUMAX-IL15, an anti-IL15 antibody developed byGenmab and Amgen, HUMAX-Inflam, developed by Genmab and Medarex,HUMAX-Cancer, an anti-Heparanase I antibody developed by Genmab andMedarex and Oxford GlycoSciences, HUMAX-Lymphoma, developed by Genmaband Amgen, HUMAX-TAC, developed by Genmab, IDEC-131, and anti-CD40Lantibody developed by IDEC Pharmaceuticals, IDEC-151 (Clenoliximab), ananti-CD4 antibody developed by IDEC Pharmaceuticals, IDEC-114, ananti-CD80 antibody developed by IDEC Pharmaceuticals, IDEC-152, ananti-CD23 developed by IDEC Pharmaceuticals, anti-macrophage migrationfactor (MIF) antibodies developed by IDEC Pharmaceuticals, BEC2, ananti-idiotypic antibody developed by Imclone, IMC-1C11, an anti-KDRantibody developed by Imclone, DC101, an anti-flk-1 antibody developedby Imclone, anti-VE cadherin antibodies developed by Imclone, CEA-CIDE(labetuzumab), an anti-carcinoembryonic antigen (CEA) antibody developedby Immunomedics, Yervoy (ipilimumab), an anti-CTLA4 antibody developedby Bristol-Myers Sequibb in the treatment of melanoma, LYMPHOCIDE(Epratuzumab), an anti-CD22 antibody developed by Immunomedics,AFP-Cide, developed by Immunomedics, MyelomaCide, developed byImmunomedics, LkoCide, developed by Immunomedics, ProstaCide, developedby Immunomedics, MDX-010, an anti-CTLA4 antibody developed by Medarex,MDX-060, an anti-CD30 antibody developed by Medarex, MDX-070 developedby Medarex, MDX-018 developed by Medarex, OSIDEM (IDM-1), and anti-Her2antibody developed by Medarex and Immuno-Designed Molecules, HUMAX®-CD4,an anti-CD4 antibody developed by Medarex and Genmab, HuMax-IL15, ananti-IL15 antibody developed by Medarex and Genmab, CNTO 148, ananti-TNFα antibody developed by Medarex and Centocor/J&J, CNTO 1275, ananti-cytokine antibody developed by Centocor/J&J, MOR101 and MOR102,anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodiesdeveloped by MorphoSys, MOR201, an anti-fibroblast growth factorreceptor 3 (FGFR-3) antibody developed by MorphoSys, tremelimumab, ananti-CTLA-4 antibody developed by Pfizer, visilizumab, an anti-CD3antibody developed by Protein Design Labs, HUZAF, an anti-gammainterferon antibody developed by Protein Design Labs, Anti-a 5β1Integrin, developed by Protein Design Labs, anti-IL-12, developed byProtein Design Labs, ING-1, an anti-Ep-CAM antibody developed by Xoma,XOLAIR® (Omalizumab) a humanized anti-IgE antibody developed byGenentech and Novartis, and MLN01, an anti-Beta2 integrin antibodydeveloped by Xoma; all of the above-cited antibody references in thisparagraph are expressly incorporated herein by reference. The sequencesfor the above antibodies can be obtained from publicly availabledatabases, patents, or literature references.

1. Exemplary Targeting Moieties

The following section provides a non-limiting list and description ofexemplary targeting moieties and their use in binding fusion proteins.

Anti-Her2:

In one embodiment, the invention provides an isolated anti-Her2 bindingfusion protein. “Anti-Her2” means a targeting moiety that specificallybinds to the extracellular domain of the HER2/neu receptor (a.k.a.erbB-2 protein), including antibodies, antibody fragments, fragmentdimers, traps, and other polypeptides with binding affinity to thedomain IV of the HER2/erbB-2 protein. The HER2-encoding gene is found onband q21 of chromosome 17, generates a messenger RNA (MRNA) of 4.8 kb,and the protein encoded by the HER2 gene is 185,000 Daltons. In normalsubject, ligands that bind to the HER2 receptor promote dimerizationwith other receptors, resulting in signal transduction and activation ofthe PI3K/Akt pathway and the MAPK pathway.

In approximately 25% of breast cancers, the HER2 gene is amplified by2-fold to greater than 20-fold in each tumor cell nucleus relative tothe number of copies of chromosome 17. Amplification of the HER2genedrives protein expression and the resulting increase in the number ofreceptors at the tumor-cell surface promotes receptor activation,leading to signaling, excessive cellular division, and the formation oftumors (Hicks, D G et al., HER2+ breast cancer: review of biologicrelevance and optimal use of diagnostic tools. Am J Clin Pathol. (2008)129(2):263-73).

The anti-Her2 used as a fusion partner with XTEN creates a bindingfusion protein composition that has can have therapeutic utility whenadministered to a subject by binding to the extracellular domain of theextracellular segment of the HER2/neu receptor. Such binding caninterfere with receptor dimerization and the resulting activation ofEGFR intrinsic tyrosine kinase function (Yarden et al, Biochemistry,(1988), 27, 3114-3118; Schlessinger, Biochemistry, (1988), 27,3119-3123), with the result that cells with bound receptors undergoarrest during the G1 phase of the cell cycle so there is reducedproliferation of tumor cells, as well as suppression of angiogenesis.

One object of the invention is to provide novel anti-Her2 binding fusionproteins comprising one or more binding moieties that specifically bindto erbB-2 protein and that do not substantially bind to normal humancells, which may be utilized for the treatment or prevention of erbB-2expressing tumor cells, or for the immunological detection of erbB-2expressing tumor cells. The CDR and FR residues of a humanized HER2antibody have been reported in Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992). In one embodiment, the anti-Her2 antibodycompositions comprise a single anti-Her2 targeting moiety linked to atleast a first XTEN. In another embodiment, the anti-Her2 compositionscomprise a first and a second anti-Her2 targeting moiety, which may bethe same or which may bind different epitopes of the erbB-2 protein. Inone embodiment, the anti-Her2 component of a binding fusion proteincomprises one or more complementarity determining regions (CDRs) oftrastuzumab capable of binding to the domain IV of the extracellularsegment of the HER2/neu receptor.

Another embodiment of the invention relates to a method of inhibitinggrowth of tumor cells by administering to a patient a therapeuticallyeffective amount of anti-Her2 binding fusion protein composition capableof inhibiting the HER2 receptor function. A further embodiment of theinvention relates to administering a therapeutically effective amount ofanti-Her2 composition capable of inhibiting growth factor receptorfunction, and a therapeutically effective amount of a cytotoxic factor.Still another object of the invention is to provide methods for thetreatment and/or prevention of erbB-2 receptor over-expressing tumorscomprising the administration of an anti-tumor effective amount of atleast one of the disclosed anti-Her2 fusion proteins capable of bindingto cancer cells associated by the over-expression of erbB-2 protein. Inanother embodiment, the invention provides a method for the treatmentand/or prevention of erbB-2 receptor over-expressing tumors comprisingthe administration of therapeutically-effective amounts of anti-Her2fusion protein comprising a first and a second anti-Her2 binding moiety,which may be the same or which may bind different epitopes of the erbB-2protein, capable of inhibiting the HER2 receptor function. Preferably,such combinations of binding moieties will exhibit better cytotoxicactivity than would be expected for the sum of the cytotoxic activity ofthe individual antibodies at the same overall antibody concentration.Additionally, one or more of the administered antibodies may beconjugated to a cytotoxic moiety, e.g., an anti-tumor drug, toxin, orradionuclide.

Anti-RSV:

In another embodiment, the invention provides an isolated anti-RSVbinding fusion protein. “Anti-RSV” means a targeting moiety thatspecifically binds to surface antigens of respiratory syncytial virus(RSV). An anti-RSV can be an antibody or fragment thereof thatneutralizes RSV, preventing its ability to establish an infection in amammal, or that contributes to the clearance of RSV from an infectedhost. The anti-RSV can be used as a fusion partner with XTEN to create afusion protein composition that has prophylactic or therapeutic utilitywhen administered to a subject, such as an infant at risk for RSVinfection. In one embodiment, the anti-RSV component of a binding fusionprotein comprises one or more complementarity determining regions (CDRs)of palivizumab. Antibodies to RSV have been described in U.S. Pat. No.5,824,307.

Anti-cMet:

In another embodiment, the invention provides an isolated anti-cMetbinding fusion protein. “Anti-cMet” means a targeting moiety thatspecifically binds to Met, or hepatocyte growth factor (HGF) receptor.MET is a proto-oncogene, with the encoded hepatocyte growth factorreceptor (HGFR) or cMet having tyrosine-kinase activity essential forembryonic development and wound healing. Upon HGF binding andstimulation, MET induces several biological responses that collectivelygive rise to invasive growth. Abnormal MET activation in cancercorrelates with poor prognosis, where aberrantly active MET triggerstumor growth, angiogenesis and formation of new blood vessels thatsupply the tumor with nutrients, and cancer spread to other organs(metastasis). MET is deregulated in many types of human malignancies,including cancers of kidney, liver, stomach, breast, and brain.Anti-cMET can be an targeting moiety that specifically binds to a HGFreceptor, serving as an antagonist to HGF. The anti-cMET can be used asa fusion partner with XTEN to create a fusion protein composition thathas prophylactic or therapeutic utility when administered to a subjectfor the treatment of MET-expressing tumors. In one embodiment, theanti-cMET component of a binding fusion protein comprises one or morecomplementarity determining regions (CDRs) of the antibody MetMab orPRO143966. Antibodies to cMet and their sequences have been described inU.S. Pat. No. 5,686,292. U.S. Pat. Nos. 6,468,529, 7,476,724 and U.S.Patent Application Publication No. 20070092520.

Anti-IL6R:

In another embodiment, the invention provides an isolated anti-IL6Rbinding fusion protein. “Anti-IL6R” means a targeting moiety thatspecifically binds to an IL-6 receptor. Anti-IL6R can serve as anantagonist to IL-6. The anti-IL6R can be used as a fusion partner withXTEN to create a fusion protein composition that has prophylactic ortherapeutic utility when administered to a subject for inflammatoryconditions, such as arthritis or Crohn's disease. Tocilizuma has beenshown to have clinical utility in moderate to severe rheumatoidarthritis, and has been approved by the FDA. In one embodiment, theanti-IL6R component of a binding fusion protein comprises one or morecomplementarity determining regions (CDRs) of tocilizuma. Antibodies toIL-6R have been described in U.S. Pat. Nos. 5,670,373, 5,795,965,5,817,790, and 7,479,543.

Anti-IL17:

In another embodiment, the invention provides an isolated anti-IL17binding fusion protein. “Anti-IL17” means a targeting moiety thatspecifically binds to the cytokine IL-17. IL-17 is a disulfide-linkedhomodimeric cytokine of about 32 kDa which is synthesized and secretedonly by CD4+ activated memory T cells (reviewed in Fossiez et al., Int.Rev. Immunol., 16: 541-551 (1998)). Interleukin (IL-17) is apro-inflammatory T cell cytokine that is expressed, for example, in thesynovial fluid of patients with rheumatoid arthritis. IL-17 is a potentinducer of various cytokines such as TNF and IL-1, and IL-17 has beenshown to have additive or even synergistic effects with TNF and IL-1.The anti-IL17 can be used as a fusion partner with XTEN to create abinding fusion protein composition that has prophylactic or therapeuticutility when administered to a subject for inflammatory conditions, suchas arthritis or Crohn's disease, or in multiple sclerosis. LY2439821 isan antibody that has shown utility, when added to oral DMARDs, inimproving signs and symptoms of rheumatoid arthritis. In one embodiment,the anti-IL6R component of a binding fusion protein comprises one ormore complementarity determining regions (CDRs) of LY2439821. Anti-IL17antibodies have been described in US Patent Application Nos. 20050147609and 20080269467 and PCT application publication WO 2007/070750.

IL17R:

In another embodiment, the invention provides an isolated IL17R bindingfusion protein. “IL17R” means a targeting moiety that specifically bindsto the cytokine receptor for IL-17. Studies have shown that contacting Tcells with a soluble form of the IL-17 receptor polypeptide inhibited Tcell proliferation and IL-2 production induced by PHA, concanavalin Aand anti-TCR monoclonal antibody (Yao et al., J. Immunol., 155:5483-5486[1995]). As interleukin (IL-17) is a pro-inflammatory T cell cytokinethat is a potent inducer of various cytokines such as TNF and IL-1, theIL17R can be used as a fusion partner with XTEN to create a bindingfusion protein composition to bind and neutralize IL-17. The IL17R canhave therapeutic utility when administered to a subject for inflammatoryconditions, such as rheumatoid arthritis or Crohn's disease. IL7Rreceptors and homologs have been cloned, as described in U.S. Pat. No.5,869,286.

Anti-IL12:

In another embodiment, the invention provides an isolated anti-IL12binding fusion protein. “Anti-IL12” means a targeting moiety thatspecifically binds to the cytokine IL-12 and, in some cases, IL-23.Biologically active IL-12 exists as a heterodimer comprised of 2covalently linked subunits of 35 (p35) and 40 (p40) kD, the latter beingknown as IL-23. IL-12 is a cytokine that is an important part of theinflammatory response, and stimulates the production of interferon-gamma(IFN-γ) and tumor necrosis factor-alpha (TNF-α) from T and naturalkiller (NK) cells, and reduces IL-4 mediated suppression of IFN-γ. Tcells that produce IL-12 have a coreceptor, CD30, which is associatedwith IL-12 activity. IL-12 has also been linked with autoimmunity andwith psoriasis, with the interaction between T lymphocytes and stem cellkeratinocytes that produce IL-12 being of significance. Ustekinumab isan anti-IL12/23 antibody that has demonstrated utility in the treatmentof moderate to severe plaque psoriasis, and has been approved by theFDA. The anti-IL-12 can be used as a fusion partner with XTEN to createa fusion protein composition that has therapeutic utility whenadministered to a subject suffering from inflammatory conditions, suchas, but not limited to, psoriasis, rheumatoid arthritis or Crohn'sdisease. In one embodiment, the anti-IL12 component of a binding fusionprotein comprises one or more complementarity determining regions (CDRs)of the antibody ustekinumab. Antibodies to IL-12 and their use have beendescribed in U.S. Pat. No. 7,279,157.

Anti-IL23:

In another embodiment, the invention provides an isolated anti-IL23binding fusion protein. “Anti-IL23” means a targeting moiety thatspecifically binds to the cytokine IL-23. IL-23 is the name given to afactor that is composed of the p40 subunit of IL-12, and is apro-inflammatory cytokine that is an important part of the inflammatoryresponse against infection. IL-23 promotes upregulation of the matrixmetalloprotease MMP9, increases angiogenesis and reduces CD8+ T-cellinfiltration. IL-23 has been demonstrated to play a role in psoriasis,multiple sclerosis and inflammatory bowel. Ustekinumab is an anti-IL23antibody that has demonstrated utility in psoriasis. The anti-IL-23 canbe used as a fusion partner with XTEN to create a fusion proteincomposition that has therapeutic utility when administered to a subjectsuffering from inflammatory conditions, such as, but not limited to,psoriasis, rheumatoid arthritis or Crohn's disease. In one embodiment,the anti-IL23 component of a binding fusion protein comprises one ormore complementarity determining regions (CDRs) of the antibodyustekinumab. Antibodies to IL-23 have been described in U.S. Pat. Nos.7,491,391 and 7,247,711.

Anti-RANKL:

In another embodiment, the invention provides an isolated anti-RANKLbinding fusion protein. “Anti-RANKL” means a targeting moiety thatspecifically binds to the protein RANKL (receptor activator of nuclearfactor kappa B Ligand or RANK ligand). RANKL is a protein that acts asthe primary signal to promote bone removal, driven by osteoclasts, whichbreak bone down. In many bone loss conditions, RANKL overwhelms thebody's natural defense against bone destruction. The anti-RANKL can beused as a fusion partner with XTEN to create a fusion proteincomposition that has therapeutic utility when administered to a subjectby inhibiting the maturation of osteoclasts by binding to RANKL,protecting the bone from degradation and thus from osteoporosis. Thebinding fusion protein therefore mimics the endogenous effects ofosteoprotegerin, another protein produced by osteoblasts which acts asan alternate receptor for RANKL, modulating the RANK/RANKL inducedosteoclast activity. Antibodies to RANKL, such as denosumab, havedemonstrated efficacy in Phase III trials demonstrated inpost-menopausal osteoporosis. In one embodiment, the anti-RANKLcomponent of a binding fusion protein comprises one or morecomplementarity determining regions (CDRs) of the antibody denosumab.Anti-RANKL antibodies have been described in U.S. Pat. No. 7,411,050.

CTLA4:

In another embodiment, the invention provides an isolated CTLA4 bindingfusion protein. “CTLA4” means a targeting moiety that specifically bindsto CD80 and CD86 on antigen-presenting cells, and can specifically bindB7. The CTLA4 can be used as a fusion partner with XTEN to create afusion protein composition that has therapeutic utility whenadministered to a subject suffering from inflammatory conditions, suchas, but not limited to, rheumatoid arthritis, psoriasis and in organtransplantation. Belatacept is a fusion protein composed of the Fcfragment of a human IgG1 immunoglobulin linked to the extracellulardomain of CTLA-4 that has shown efficacy in providing extended graftsurvival. In one embodiment, the CTLA4 binding component of the bindingfusion protein comprises one or more binding regions from belatacept.The cloning and use of CTLA4 compositions have been described in U.S.Pat. Nos. 5,434,131, 5,773,253, 5,851,795, 5,885,579, 7,094,874, and7,439,230.

ANTI-CD3:

In another embodiment, the invention provides an isolated anti-CD3binding fusion protein. “Anti-CD3” means a targeting moiety thatspecifically binds to CD3 T-cell receptor. T-Cell CoReceptor is aprotein complex composed of four distinct chains; a CD3γ chain, a CD3δchain, and two CD3ε chains. These chains associate with a molecule knownas the T cell receptor (TCR) and the chain to generate an activationsignal in T lymphocytes. Anti-CD3 monoclonal antibodies suppress immuneresponses by transient T-cell depletion and antigenic modulation of theCD3/T-cell receptor complex. For example, anti-CD3 treatment of adultnonobese diabetic (NOD) mice, a spontaneous model of T-cell-mediatedautoimmune insulin-dependent diabetes mellitus, can inhibit theautoimmune process leading to diabetes. The use of anti-CD3 antibodiesto treat diseases and disorders has been described, for example, in U.S.Pat. No. 4,515,893. In one embodiment, the CD3 binding component of thebinding fusion protein comprises one or more complementarity determiningregions (CDRs) of the antibody Muromonab-CD3.

ANTI-CD40:

In another embodiment, the invention provides an isolated anti-CD40binding fusion protein. “Anti-CD40” means a targeting moiety thatspecifically binds to the cell-surface receptor CD-40. CD-40 is acell-surface receptor that plays a role in immune responses, as well ascell growth and survival signaling when activated by CD40 ligand(CD40L). CD40 is commonly over-expressed and activated in B-cellmalignancies, such as multiple myeloma and lymphoma. The anti-CD40 canbe used as a fusion partner with XTEN to create a fusion proteincomposition that can have therapeutic utility when administered to asubject suffering from various cancers, particularly B-cellmalignancies. In one embodiment, the anti-CD40 component of a bindingfusion protein comprises one or more complementarity determining regions(CDRs) of the antibody lucatumumab. Anti-CD40 antibodies have beendescribed in U.S. Pat. No. 7,445,780, and U.S. Patent Appl. Nos.20070110754 and 20080254026.

ANTI-TNFalpha:

In another embodiment, the invention provides an isolated anti-TNFalphabinding fusion protein. “Anti-TNFalpha” means a targeting moiety thatspecifically binds to the cytokine TNFalpha. TNFalpha, or cachexin, is apro-inflammatory cytokine involved in systemic inflammation and is amember of a group of cytokines that stimulate the acute phase reaction.The primary role of TNF is in the regulation of immune cells. TNF isproduced mainly by macrophages, but is also produced by lymphoid cells,mast cells, endothelial cells, cardiac myocytes, adipose tissue,fibroblasts, and neuronal tissue. Large amounts of TNF are released inresponse to lipopolysaccharide and Interleukin-1 (IL-1). TNF has beenimplicated in autoimmune disorders such as rheumatoid arthritis,ankylosing spondylitis, Crohn's disease, psoriasis and refractoryasthma, and plays a role in septic shock and other serious forms ofacute inflammatory response and SIRS. The anti-IL-TNFalpha can be usedas a fusion partner with XTEN to create a fusion protein compositionthat can have therapeutic utility in a wide variety of inflammatorydisorders, including rheumatoid arthritis, ankylosing spondylitis,Crohn's disease, psoriasis and refractory asthma. Anti-TNFalphaantibodies, such as infliximab and etanercept have shown efficacy inpsoriasis, Crohn's disease, ankylosing spondylitis, psoriatic arthritis,rheumatoid arthritis and ulcerative colitis. In one embodiment, theanti-TNFalpha component of a binding fusion protein comprises one ormore complementarity determining regions (CDRs) or binding regions ofthe infliximab or etanercept. Anti-TNF antibodies have been described inU.S. Pat. No. 6,790,444, and chimeric antibodies comprising a TNFreceptor have been described in U.S. Pat. No. 5,605,690.

The invention provides binding fusion protein compositions in which thebinding regions of the foregoing referenced exemplary targeting moietiesare sequence variants. For example, it will be appreciated that variousamino acid deletions, insertions and substitutions can be made in thetargeting moiety to create variants without departing from the spirit ofthe invention with respect to the binding activity or the pharmacologicproperties of the binding fusion protein. Examples of conservativesubstitutions for amino acids in polypeptide sequences are shown inTable 5. However, in embodiments of the binding fusion protein in whichthe sequence identity of the targeting moiety is less than 100% comparedto a specific sequence referenced or disclosed herein, the inventioncontemplates substitution of any of the other 19 natural L-amino acidsfor a given amino acid residue of the given targeting moiety, which maybe at any position within the sequence of the targeting moiety orbinding region of the targeting moiety, including adjacent amino acidresidues. If any one substitution results in an undesirable change inbinding activity, then one of the alternative amino acids can beemployed and the construct protein evaluated by the methods describedherein (e.g., the assays of the Examples), or using any of thetechniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934, thecontents of which is incorporated by reference in its entirety, or usingmethods generally known in the art. In addition, variants can include,for instance, polypeptides wherein one or more amino acid residues areadded or deleted at the N- or C-terminus of the referenced or disclosedamino acid sequence of a targeting moiety that retains some if not allof the binding activity of the referenced or disclosed targeting moiety;e.g., the ability to bind a target of Table 1 or Table 2.

TABLE 5 Exemplary conservative amino acid substitutions Original ResidueExemplary Substitutions Ala (A) val; leu; ile Arg (R) Lys; Gln; Asn Asn(N) Gln; His; Llys; Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) AspGly (G) Pro His (H) Asn; Gln; Lys; Arg Ile (I) Leu; Val; Met; Ala; Phe;Norleucine Leu (L) Norleucine; Ile: Val; Met; Ala: Phe Lys (K) Arg' Gln;Asn Met (M) Leu; Phe; Ile Phe (F) Leu; Val; i = Lle; Ala Pro (P) Gly Ser(S) Thr Thr (T) Ser Trp (W) Tyr Tyr(Y) Trp; Phe: Thr; Ser Val (V) Ile;Leu; Met; Phe; Ala; Norleucine

2. Exemplary Forms of Targeting Moieties

The following section provides a non-limiting list and description ofexemplary forms of targeting moieties.

“Antibody” or “antibodies”, as used here, refers to a targeting moietyconsisting of one or more polypeptides substantially encoded byimmunoglobulin genes or fragments of immunoglobulin genes, and is usedin the broadest sense to cover intact monoclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies or fragment thereof, and antibody fragmentsso long as they exhibit the desired biological activity; e.g., bindingaffinity to a target ligand or antigen.

Immunoglobulins can be assigned to different classes depending on theamino acid sequence of the constant domain of their heavy chains. Thereare five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM,and several of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

The term “monoclonal” indicates the character of the targeting moietyantibody or antibody fragment as being obtained from a substantiallyhomogeneous population of antibodies or fragments, and is not to beinterpreted as requiring production of the antibody by a particularmethod. For example, while the monoclonal antibodies created inaccordance with the methods of the present invention may be made by thehybridoma method first described by Kohler et al., Nature, 256:495(1975), they may also be synthetics made by recombinant DNA methods(see, e.g., U.S. Pat. No. 4,816,567) and expressed in either mammalianor non-mammalian hosts; e.g., E. coli. The substitution of immortalizedcells with bacterial cells considerably simplifies procedures forpreparing large amounts of the inventive binding fusion proteinmolecules. Furthermore, a recombinant production system allows theability to produce tailor-made antibodies and fragments thereof, or evenlibraries to screen for specific attributes. For example, it is possibleto produce chimeric molecules with new combinations of binding andeffector functions, humanized antibodies and novel antigen-bindingmolecules, including bifunctional binding fusion proteins. Furthermore,the use of polymerase chain reaction (PCR) amplification (Saiki, R. K.,et al., Science 239, 487-491 (1988)) to introduce variations into thesequence and isolate antibody producing sequences from cells has greatpotential for speeding up the timescale under which specificities can beisolated. Amplified V_(H) and V_(L) genes can be cloned directly intovectors for expression in bacteria or mammalian cells (Orlandi, R., etal., 1989, Proc. Natl. Acad. Sci., USA 86, 3833-3837; Ward, E. S., etal., 1989 supra; Larrick, J. W., et al., 1989, Biochem. Biophys. Res.Commun. 160, 1250-1255; Sastry, L. et al., 1989, Proc. Natl. Acad. Sci.,USA, 86, 5728-5732). Soluble antibody fragments secreted from bacteriacan then be screened in binding assays described herein, or others knownin the art, to select those constructs with binding activitiessufficient to meet the application.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or has a high degree of homology to correspondingparental sequences in antibodies derived from a particular firstspecies, while the remainder of the chain(s) is identical with or has ahigh degree of homology to sequences in antibodies derived from a secondspecies, wherein the resulting antibody exhibits the desired biologicalactivity; e.g., binding affinity for the target antigen or ligand (U.S.Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-4855 (1984)).

The term “humanized” means forms of antibodies, including fragments,that are chimeric in that they include minimal sequence derived fromnon-human immunoglobulin but otherwise comprise sequence from humanimmunoglobulins. Methods for humanizing non-human antibodies have beendescribed in the art. Preferably, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human(e.g., murine, rat, or non-human primate) and that are typically takenfrom a variable domain of a V_(L) or V_(H) chain having the desiredspecificity and affinity for the target ligand. Humanization can beessentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting hypervariable region sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (see, e.g., U.S. Pat. No. 4,816,567) wherein allor a portion of the human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some hypervariableCDR residues and possibly some FR residues are substituted by residuesfrom analogous sites in rodent (or other non-human species, e.g.,non-human primates) antibodies. In one embodiment, humanized antibodiescomprise residues that are not found in the recipient antibody or in thedonor antibody to, for example, increase binding affinity or some otherproperty. In general, humanized antibodies comprise substantially all ofat least one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to or havesequences derived from those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence. The humanized antibody can optionally comprise at least aportion of an immunoglobulin constant region (Fc), preferably that of ahuman immunoglobulin.

The targeting moieties of the subject compositions can be derived fromhumanized antibodies. The choice of human variable domains, both lightand heavy, to be used in the compositions is very important to reduceantigenicity of the antibody. For example, the sequence of the variabledomain of a rodent antibody can be screened against a library of knownhuman variable-domain sequences in order to select a sequence that isless likely to elicit an immune response in the recipient. In acorresponding fashion, the human sequence that is closest to that of therodent can be used as the human framework (FR) for the humanizedantibody (Sims et al., J. Immunol, 151:2296 (1993); Chothia et al., J.Mol. Biol., 196:901 (1987)). The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immnol., 151:2623 (1993)).

An additional property is that targeting moieties can be humanized yetretain high affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized targeting moieties are prepared by an iterative process ofanalysis of the parental sequences and various conceptual humanizedproducts using three-dimensional models of the parental and humanizedsequences followed by testing. Three-dimensional immunoglobulin modelsare commonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the recipient anddonor using standard recombinant DNA techniques so that the desiredcharacteristic, such as increased affinity for the target antigen(s),can be achieved. In one embodiment, binding fusion protein constructsare created in which a sequence comprising linked heavy chain variabledomains is linked to a heavy chain constant domain and sequencecomprising linked light chain variable domains is linked to a lightchain constant domain. Preferably the constant domains are human heavychain constant domain and human light chain constant domainrespectively. In a further embodiment of the foregoing, the fusionprotein can be designed to include portions or all of a hinge region inorder to permit dimerization of the binding fusion protein, and whichcan further comprise an XTEN linked to the C-terminus of the constantregion. In an alternative embodiment, the binding fusion protein can bedesigned to incorporate a partial Fc without a hinge and with a CH2domain that is truncated but retains FcRn binding in order to conferlonger terminal half-life on the construct. In yet another embodiment,the binding fusion protein can be designed to incorporate a partial Fcwithout hinge but with a CH2 and CH3 domain, which can dimerize via theCH3 domain. In the embodiments hereinabove described in this paragraph,an XTEN can be linked to either the N- or C-terminus of the fusionprotein, to enhance one or more properties of the resulting bind fusionprotein.

“Antibody fragments” comprise a portion of an intact antibody or asynthetic or chimeric counterpart, preferably the antigen binding orvariable region of the intact antibody. Examples of antibody fragmentsinclude molecules such as Fab fragments, Fab′ fragments, F(ab′)₂fragments, Fd fragments, Fabc fragments, Fd fragments, Fabc fragments,domain antibodies (V_(HH)), single-chain antibody molecules (scFv),diabodies, individual antibody light chains, individual antibody heavychains, chimeric fusions between antibody chains and other molecules,and the like.

A “Fab fragment” refers to a region of an antibody which binds toantigens. A Fab fragment is composed of one constant and one variabledomain of each of the heavy and the light chain. These domains shape theparatope—the antigen binding site—at the amino terminal end of themonomer. The two variable domains bind the epitope on their specificantigens. A Fab fragment can be linked by a disulfide bond at theC-terminus. Fab fragments can be generated in vitro. For example, theenzyme papain can be used to cleave an immunoglobulin monomer into twoFab fragments and an Fc fragment. The enzyme pepsin cleaves below thehinge region, so a F(ab′)₂ fragment and a Fc fragment is formed. Asdescribed more fully below, variable regions of the heavy and lightchains can be fused together to form a single chain variable fragment(scFv), which retains the original specificity of the parentimmunoglobulin.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains.

The term “variable” refers to the fact that portions of the variabledomains differ extensively in sequence among antibodies and confer thebinding specificity of each particular antibody for its particularantigen. The variability is concentrated in three segments calledcomplementarity-determining regions (CDRs) or hypervariable regions,both in the light-chain and the heavy-chain variable domains; i.e.,CDR1, CDR2 and CDR3. In particular, the CDR regions from antibodies canbe incorporated into targeting moieties of the subject compositions, butcan be individually selected from one or more antibodies to create thebinding domain. The more highly conserved portions of variable domainsare called the framework regions (FR), which may also be incorporatedinto targeting moieties. The variable domains of native heavy and lightchains each comprise four FR regions, typically adopting a β-sheetconfiguration, connected by three CDRs that form loops. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., NIH Publ. No.91-3242, Vol. I, pages 647-669 (1991)). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibit orparticipate in various effector functions, such as antibody-dependentcellular toxicity.

Single-Chain Variable Fragment Binding Fusion Proteins

In one aspect, the present invention provides single-chain variablefragment binding fusion protein compositions. The term “single-chainvariable fragment” or “scFv” means an antibody fragment that comprisesone V_(H) and one V_(L) domain of an antibody, wherein these domains arepresent in a single polypeptide chain, and are generally joined by apolypeptide linker between the domains that enables the scFv to form thedesired structure for antigen binding. Methods for making scFv's areknown in the art (see, e.g., U.S. Pat. No. 6,806,079; Bird et al. (1988)Science 242:423-426; Huston et al. (1988) PNAS 85:5879-5883; Pluckthunin The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg andMoore eds. Springer-Verlag, New York, pp. 269-315 (1994)).

A binding domain of the scFv binding fusion protein compositions of theinvention can have the N- to C-terminus configuration VH-linker-VL orVL-linker-VH. The binding fusion proteins would include at least a firstXTEN and optionally a second XTEN sequence linked to the N- orC-terminus of the fusion protein (as shown in FIG. 1A-FIG. 1B),resulting in at least the following structure permutations (N- toC-terminus); XTEN-VH-linker-VL; VH-linker-VL-XTEN;XTEN-VH-linker-VL-XTEN; XTEN-VL-linker-VH; VL-linker-VH-XTEN;XTEN-VL-linker-VH-XTEN. In one embodiment of the foregoing, thecomposition comprises an XTEN linked to the N-terminus of the fusionprotein, wherein the expression of the fusion protein in a host celltransformed with a suitable expression vector comprising apolynucleotide encoding the fusion protein comprising the N-terminalsequence is enhanced compared to the expression of a correspondingfusion protein from a polynucleotide not comprising the N-terminal XTENencoding sequence. In such cases, the N-terminal XTEN could either havejust the short sequence that enhances expression, or could furtherinclude a long XTEN of at least greater than 400 to about 3000 aminoacid residues between the N-terminal piece and a binding domain toconfer enhanced pharmacokinetic or pharmaceutical properties to thefusion protein, as described above. In one embodiment of the foregoing,the N-terminal XTEN sequence comprises a sequence that exhibits at leastabout 80% sequence identity, or 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a sequence selected from AE48, AM48, AE624, AE913, andAM923. In another embodiment of the fusion proteins, the linkers, theN-terminal XTEN, as well as the long carrier XTEN can comprise asequence that can be a fragment of or that exhibits at least about 80%sequence identity, or 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto a sequence selected from any one of Tables 4 or 11-15.

The linkers utilized to join the components of the binding fusionproteins should be flexible in nature. In one embodiment the linkerjoining the V_(L) and V_(H) binding domains that form the antigenbinding site of the scFv targeting moiety can have from about 15 toabout 30 amino acid residues in length. In another embodiment, thelinker can have from about 30 to about 200 amino acid residues, or about40 to about 144 amino acid residues, or about 50 to about 96 amino acidresidues. In any of the embodiments hereinabove described in thisparagraph, the linker can be a sequence derived from a fragment of anyof the XTEN sequences of Tables 4 or 11-15. In another embodiment, thelinker can be a sequence in which at least 80% of the residues arecomprised of amino acids glycine, serine, and/or glutamate, such as, butnot limited to a sequence with about 80-100% sequence identify to thesequence GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 1), or a portion ora multimer thereof.

In one embodiment, the invention provides binding fusion proteinscomprising two or more scFv targeting moieties (as shown in FIG. 2A-FIG.2B). In one embodiment, the two or more scFv targeting moieties may beidentical. In another embodiment, the two or more scFv targetingmoieties may be different and may bind to different targets (e.g., twoor more targets of Table 1) or to different epitopes on the same target.In the foregoing embodiments, the two or more scFv targeting moietiescan be joined by a linker sequence. The invention contemplates that thelinkers as well as the long carrier XTEN and the N-terminal XTEN cancomprise a sequence that can be a fragment of or that exhibits at leastabout 80% sequence identity, or 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a sequence selected from any one of Tables 4 or 11-15.

The invention contemplates various configurations of the multivalentscFv-XTEN binding fusion proteins, with the two or more targetingmoieties, linkers and one or more XTEN in various N- to C-terminusconfiguration; e.g., TM1-L-TM2-XTEN, XTEN-TM1-L-TM2,XTEN-TM1-L-TM2-XTEN, etc.

The general methodology for the assembly of the components, theexpression and recovery, followed by characterization of the bindingfusion protein is illustrated in FIG. 9.

Diabody Binding Fusion Proteins

In another aspect, the invention provides compositions of diabodybinding fusion proteins. The term “diabody” or “diabodies”, as usedherein, refers to fusion proteins comprising antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) by ashort linker in the same polypeptide chain. In one embodiment, thediabody binding fusion protein composition is a single polypeptide chainhaving two binding moieties, each with V_(L) and V_(H) interconnected bya flexible linker of about 30 to about 300 amino acid residues joiningthe C-terminus of one binding domain pair with the N-terminus of thesecond binding domain pair, and one or more XTEN on the N- and/orC-terminus (as shown in FIG. 3A-FIG. 3B). In this case, the compositionis created by selective incorporation of a linker that is too short toallow pairing between the two adjacent domains, but the targeting moietyregions are connected by longer flexible linker to permit theassociation between the binding domains from the opposite ends of thefusion protein. For example, it is known that a scFv molecule with alinker 3-12 residues long cannot fold into a functional Fv domain but,generally, instead associates with a second scFv molecule to form amultivalent diabody with two binding sites, while a linker of 1-2residues tends to favor triabody formation with three binding sites(John L. Atwell et al., Protein Engineering (1999) 12(7):597-604). Thus,in some embodiments, the invention provides diabody binding fusionproteins in which the adjacent binding domain pairs are linked by 1-2 or3-12 amino acid residues and the binding domain pairs are, in turn,linked by a flexible XTEN linker. In one embodiment of the foregoing,the flexible linker can be an XTEN sequence of about 100 to about 300amino acid residues. In another embodiment the flexible linker sequencecan be comprised of amino acids such as glycine, serine, and/orglutamate making up about 80-100% of the sequence. In anotherembodiment, the linker can be a sequence in which at least 80% of theresidues are comprised of amino acids glycine, serine, and/or glutamate,such as, but not limited to a sequence with about 80-100% sequenceidentify to the sequence GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 1),or a portion or a multimer thereof.

In one embodiment, the invention provides a diabody binding fusionprotein that can be monospecific, binding one type of antigen. Inanother embodiment, the invention provides a bispecific diabody bindingfusion protein in which the targeting moieties are directed to differentantigens or different epitopes on the same antigen. In the foregoingembodiments, the specificity of the diabody binding fusion is determinedby the incorporation of either identical or different respective V_(H)and V_(L) components.

An illustration of one example of a diabody binding fusion proteins isshown in FIG. 2A-FIG. 2B, however the invention contemplates differentconfigurations of the diabody binding fusion proteins; e.g., a differentN- to C-terminus order of the V_(H) and V_(L) domains, or deleting oneof the XTEN sequences from either the N- or C-terminus.

In one embodiment of the foregoing, the composition comprises an XTENlinked to the N-terminus of the diabody binding fusion protein, whereinthe expression of the fusion protein in a host cell transformed with asuitable expression vector comprising a polynucleotide encoding thefusion protein comprising the N-terminal sequence is enhanced comparedto the expression of a corresponding fusion protein from apolynucleotide not comprising the N-terminal XTEN encoding sequence.

In addition, the invention provides multimers that are trivalent (havingthree antigen-binding sites), tetravalent (having four antigen-bindingsites), and so on. In another embodiment of the diabody fusion proteins,the linkers as well as the long carrier XTEN and the N-terminal XTEN cancomprise a sequence that can be a fragment of or that exhibits at leastabout 80% sequence identity, or 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a sequence selected from any one of Tables 4 or 11-15.

The general methodology for the assembly of the components, theexpression and recovery, followed by characterization of the bindingfusion protein is illustrated in FIG. 9.

Domain Antibody Binding Fusion Proteins

In another aspect, the invention provides single chain fusion proteinscomprising domain antibody targeting moieties linked to XTEN, whereinthe fusion protein is able to bind to an antigen or ligand. As usedherein, “domain antibody”, or “V_(HH),” is an immunoglobulin having avariable region (V_(H)) with an antigen-binding site which will aloneallows the recognition and complete binding of an antigen, linked to aconstant region (C_(H)), but are devoid of the first domain of theconstant region (C_(H)1) and are devoid of a V_(L) domain. The V_(HH) donot correspond to fragments obtained, for instance, by the degradationof a natural four-chain model immunoglobulin. V_(HH) are devoid of lightchains, such that the variable domains of their heavy chains haveproperties differing from those of the four-chain immunoglobulinvariable heavy chain (V_(H)), including no normal interaction sites withthe V_(L) or with the C_(H)1 domain. V_(HH) can adopt athree-dimensional organization that distinguishes from the conventionalthree-dimensional organization of four-chain antibodies according to thedescription that is given by Chothier C. and Lesk A. M, (1987—J. Mol.Biol. 197, 901-917). V_(HH) can comprise type G immunoglobulins,especially of class 2 (IgG2) or class 3 (IgG3). The parental V_(HH)immunoglobulin sequences can be derived from certain animals, especiallyfrom members of the camelid family, or from sharks, which can then begenerated in host cells by genetic engineering or by chemical synthesis.Appropriate host cells include bacteria (e.g. E. coli) and eukaryoticcells, such as yeasts or animal cells including mammalian cells, orplant cells.

In one embodiment, the binding fusion protein can comprise one V_(HH)targeting moiety. In another embodiment, the binding fusion protein cancomprise two or more V_(HH) targeting moieties; e.g., two, or three, orfour, or five, or six or more V_(HH) targeting moieties. In such case,the linker sequence between the V_(HH) fragments can be, for example, asequence corresponding to a fragment of the hinge domain of animmunoglobulin (e.g. the long hinge domain) or can be a short XTENsequence of about 30 to about 300 amino acid residues, or can be asequence in which at least 80% of the residues are comprised of aminoacids glycine, serine, and/or glutamate, such as, but not limited to asequence with about 80-100% sequence identify to the sequenceGSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 1), or a portion or amultimer thereof. In one embodiment of the foregoing, the V_(HH) arehetero-specific with binding affinity to different epitopes of the sameantigen. In another embodiment, the V_(HH) are hetero-specific withbinding affinity to heterologous antigens. The invention contemplatesthat the V_(HH) binding fusion protein would comprise an additional XTENsequence of at least greater than about 400 to about 3000 residues inwhich the XTEN would confer enhanced properties to the composition, asdescribed above. Thus, the invention contemplates binding fusion proteinconfigurations including, but not limited to V_(HH)—XTEN; XTEN-V_(HH);V_(HH)—Linker-V_(HH)-XTEN; XTEN-V_(HH)-Linker-V_(HH); andXTEN-V_(HH)-Linker-V_(HH)-XTEN, or multimers thereof. In the foregoing,for those configurations with two V_(HH), the V_(HH)− can be identicalor can be different, in the latter case binding two differentligands/antigens or different epitopes on the same ligand/antigen. In anembodiment of the foregoing domain binding fusion proteinconfigurations, the composition comprises an XTEN linked to theN-terminus of the fusion protein, wherein the expression of the fusionprotein in a host cell transformed with a suitable expression vectorcomprising a polynucleotide encoding the fusion protein comprising theN-terminal sequence is enhanced compared to the expression of acorresponding fusion protein from a polynucleotide not comprising theN-terminal XTEN encoding sequence.

In another embodiment of the domain fusion proteins, the linkers as wellas the long carrier XTEN and the N-terminal XTEN can comprise a sequencethat can be a fragment of or that exhibits at least about 80% sequenceidentity, or 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to asequence selected from any one of Tables 4 or 11-15. In anotherembodiment, the linker can be a sequence in which at least 80% of theresidues are comprised of amino acids glycine, serine, and/or glutamate,such as, but not limited to a sequence with about 80-100% sequenceidentify to the sequence GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 1),or a portion or a multimer thereof.

The general methodology for the assembly of the components, theexpression and recovery, followed by characterization of the bindingfusion protein is illustrated in FIG. 9.

Cytokine Binding Fusion Proteins

In another aspect, the invention provides monomeric fusion proteinscapable of binding a cytokine to form a nonfunctional complex,comprising receptor-like binding domains joined by an XTEN, wherein theresulting fusion protein has binding affinity for the target cytokineligand. As used herein, “cytokines” means a category of soluble, smallprotein signaling molecules that are used extensively in cellularcommunication, including but not limited to interleukins, interferons,chemokines, growth factors, colony stimulating factors, tumor necrosisfactors, peptide hormones, and other immunomodulating agents thatmediate a variety of biological effects, including the regulation of thegrowth and differentiation of many cell types. The action of cytokinesmay be autocrine, paracrine, and endocrine. For example, humaninterleukin-17 is a cytokine which stimulates the expression ofinterleukin-6, intracellular adhesion molecule 1, interleukin-8,granulocyte macrophage colony-stimulating factor, and prostaglandin E2expression, and plays a role in the preferential maturation of CD34+hematopoietic precursors into neutrophils (Yao et al., J. Immunol.155:5483 (1995); Fossiez et al., J. Exp. Med. 183:2593 (1996)).

Cytokines are generally bound by cell-surface receptors that, in turn,trigger cascades of intracellular signaling that alter cell functions.These may include upregulation and/or down-regulation of several genesand their transcription products, resulting in the production of othercytokines, an increase in the number of surface receptors for othermolecules, or the suppression of their own effect by feedbackinhibition. The term “receptor” denotes a cell-associated protein thatbinds to a bioactive molecule and mediates the effect of the ligand onthe cell. “Receptor”, as used herein, includes, but is not limited tocytokine receptors and chemokines receptors. While many native cellreceptors have binding domains that are immunoglobulin-like, the term“receptor” as used herein specifically excludes immunoglobulins thatconstitute antibodies or MHC receptors.

Receptors that bind cytokines are typically composed of one or moreintegral membrane proteins that have an extracellular component thatbinds the cytokine with high affinity and transduces this binding eventto the cell through the transmembrane and cytoplasmic portions of thereceptor subunits. The ligand-binding subunit of a receptor is referredto as the alpha chain, while other signal transducing subunits are namedbeta chains, or gamma chains. Thus, the ability to sequester cytokines,hormones or related ligands prior to binding to their natural cellreceptors represents a mechanism to alter the pathogenesis of diseases,disorders or conditions.

Cytokine receptors have been grouped into several families on the basisof similarities in their extracellular ligand binding domains;immunoglobulin superfamily receptor, Class I cytokine receptor family,Class II cytokine receptor family, TNF receptor family, and chemokinereceptor family. In some embodiments, the invention provides cytokinebinding moieties that comprise binding domains, portions of bindingdomains, or sequences with sequence identity to members of the foregoingreceptor families (except for antibody immunoglobulins specificallyexcluded above) linked to one or more XTEN sequences, wherein thecytokine binding moiety has at least a portion of the binding affinitycompared to the native receptor and, when ligand is sequestered, is ableto interfere with the binding of the target ligand to the nativereceptor. In another embodiment, the invention provides a receptorbinding fusion protein that comprise binding domains, portions ofbinding domains, or sequences with sequence identity to immunoglobulinsuperfamily receptors linked to one or more XTEN sequences, wherein thebinding domains have at least a portion of the binding affinity comparedto the native receptor and, when ligand is sequestered, is able tointerfere with the binding of the target ligand to the native receptor.

By “cytokine binding moiety” what is meant is at least that minimalportion of the extracellular ligand-binding domain of a cytokinereceptor necessary to bind the cytokine. For example, while somecytokine receptors have components designated a and 13, one skilled inthe art would know which component of the receptor is the signaltransducing component and which is the ligand-binding component. Thus topractice the present invention and create a high affinity bindingprotein for a cytokine, one of skill in the art would create an isolatednucleic acid comprising a nucleotide sequence encoding a first fusionpolypeptide component comprising the amino acid sequence of the cytokinebinding portion of the extracellular domain of the specificitydetermining component of the receptor; a nucleotide sequence encoding asecond fusion polypeptide component comprising the amino acid sequenceof a cytokine binding portion of the extracellular domain that may bethe same or may be different from the first, and a nucleotide sequenceencoding a third fusion polypeptide component comprising the amino acidsequence of an XTEN linking the binding components to create a geneencoding a high affinity cytokine binding fusion protein for the targetcytokine. The general methodology for the assembly of the components,the expression and recovery, followed by characterization of the bindingfusion protein is illustrated in FIG. 9.

As used herein, the term “binding domain” may itself include amulti-domain structure wherein two or more individual domains must beclosely linked to form a single binding region. Examples of theforegoing are the native receptors to VEGF, flt-1 and flk-1, both ofwhich have seven immunoglobulin-like domains in each of two arms thatform the extracellular ligand-binding regions of the receptors(Matthews, et al., PNAS 88:9026 (1991)). However, it has beendemonstrated that recombinant proteins comprising either two or three ofthe seven domains can, when paired with a corresponding second armcomprising either two or three domains, can bind VEGF with high affinity(see U.S. Pat. Nos. 5,952,199, 7,374,757). In some embodiments, VEGFcytokine binding fusion proteins comprise pairs of an Ig domain 2 (D2)and an Ig domain 3 (D3) derived from one or more VEGF receptors, asshown in FIG. 4A-FIG. 4B. In one embodiment of the foregoing, the VEGFreceptor is Flt-1. In another embodiment of the foregoing, the VEGFreceptor is Flk-1. In yet another embodiment of the foregoing, the VEGFreceptor is Flt-4. The domain units may comprise VEGF receptor domainsconnected directly to each other or, optionally, be connected viaspacers or linkers, such as fragments of XTEN.

“Immunoglobulin-like domain” or “Ig-like domain” or “ligand-bindingdomain” refers to independent and distinct domains that are found in theextracellular ligand-binding region of cytokine receptors and it isspecifically intended that the term encompass not only the completewild-type domain, but also insertional, deletional and substitutionalvariants thereof that retain at least a portion of the binding affinityof the wild-type domain. It will be readily apparent to those ofordinary skill in the art that numerous variants of the domains orcombinations of the domains of the cytokine binding proteins can beobtained which will retain substantially the same functionalcharacteristics as the wild type domain.

The invention also contemplates cytokine binding fusion proteinscomprising binding domains derived from multimeric receptors. Multimericreceptors include homodimers (e.g., PDGF receptor αα and ββ isoforms,erythropoietin receptor, MPL, and G-CSF receptor), heterodimers whosesubunits each have ligand-binding and effector domains (e.g., PDGFreceptor αβ isoform), and multimers having component subunits withdisparate functions (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, andGM-CSF receptors).

The invention contemplates a number of different constructs employingcytokine binding domains known to have affinity for target ligands ofinterest. Preferred embodiments of the invention include fusion proteincompositions capable of binding a ligand comprising at least a first anda second polypeptide binding domain operatively fused to a linkingcomponent, wherein the linking component comprises a flexible XTENsequence. In one embodiment of the foregoing, the composition furthercomprises at least a second XTEN as a carrier, wherein the carrier mayenhance the pharmacokinetic or pharmaceutical properties of the fusionprotein. In another embodiment of the foregoing, the compositioncomprises an XTEN linked to the N-terminus of the fusion protein,wherein the expression of the fusion protein in a host cell transformedwith a suitable expression vector comprising a polynucleotide encodingthe fusion protein comprising the N-terminal sequence is enhancedcompared to the expression of a corresponding fusion protein from apolynucleotide not comprising the N-terminal XTEN encoding sequence.

In a particular feature of the cytokine binding fusion proteins, theunstructured characteristic of the XTEN linker permits the respectivebinding domains to adopt a flexible configuration to freely andcorrectly orient with the ligand for optimal binding. “Flexibleconfiguration”, when used in reference to the cytokine binding proteincompositions of the present disclosure, means that the receptorpolypeptide sequences or domains of the fusion protein are not held in aparticular configuration relative to each other but are free to move,under physiologic conditions, to the extent that the XTEN polypeptidestethered to the receptor polypeptides permit. Based upon thesecharacteristics, it is believed that a binding domain of the fusionprotein is more likely to encounter and attach to the target ligand,under physiologic conditions, compared to a construct wherein thedomains are held in a fixed orientation, such as receptor traps in whichthe binding domains are fused to and held in place by the dimerizationbetween Fc domains or heavy chains of IgG (see, e.g., U.S. Pat. No.7,417,134). Not to be bound by a particular theory, upon encounteringthe target ligand under physiologic or assay conditions, the bindingdomains of the inventive fusion protein can mutually orientatesubstantially as in a native cytokine receptor, adopting a constrainedconfiguration to sequester the ligand. “Sequester” or “sequestering”when used in reference to the activity of a cytokine binding fusionprotein of the present invention means that the fusion protein binds tothe target ligand to form a substantially nonfunctional complex,interfering with its ability to bind to a native receptor. As usedherein, “substantially nonfunctional complex” means that the residualactivity of the bound ligand and/or its ability to bind to its cognatereceptor would be less than about 60%, or less than about 50%, or lessthan about 40%, or less than about 30%, or less than about 20%, or lessthan about 10%, or less than about 5%, or less than about 1% compared toun-bound ligand with a native receptor. The cytokine binding fusionproteins are thus antagonists.

In light of the foregoing, the binding fusion protein compositions canhave an “open” conformation (FIG. 4A) such that, when a target moleculeis in close proximity to the binding fusion protein, the interactionwith the domains permits a change in conformation wherein both domainunits come into association with the target ligand, creating a “closed”conformation (FIG. 4B), effectively sequestering the target molecule.

The fusion proteins of any of the foregoing cytokine binding proteinembodiments can further comprise a second and optionally a third XTENprotein, as illustrated in FIG. 4A-FIG. 4B, to impart the enhancedcharacteristics as a carrier or as a N-terminal XTEN, as describedabove.

In one embodiment, the cytokine binding proteins are multivalent fusionproteins comprising two binding region domains that are homologous, theXTEN linker, and can further comprise the additional XTEN sequences onthe N- and/or C-terminus.

In another embodiment of the cytokine binding fusion proteins, thelinkers as well as the long carrier XTEN and the N-terminal XTEN cancomprise a sequence that can be a fragment of or that exhibits at leastabout 80% sequence identity, or 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a sequence selected from any one of Tables 4 or 11-15.

Receptor Binding Fusion Proteins

In another aspect, the invention provides binding fusion proteinscomprised of at least a first binding region comprising a first andoptionally a second binding domain derived from Ig-like domains fromcell receptors linked to at least a first XTEN. A non-limiting exampleof the foregoing is a binding protein with Ig-like binding domainsderived from the VEGF receptor, with XTEN linked to either the N- orC-terminus of the binding regions, as illustrated in FIGS. 4 and 5. In aparticular embodiment of the foregoing, the binding fusion protein cancomprise two Ig-like binding regions linked by a short XTEN linker ofabout 20 to about 200 amino acid residues (e.g., a fragment of an XTENsequence of Table 4), and a longer XTEN carrier, wherein the VEGFbinding regions can bind and substantially sequester dimeric VEGF. Inanother embodiment, the binding fusion protein can comprise two sets oftwo Ig-like binding regions, which can be identical or can be differentdomains, linked by a short XTEN linker of about 20 to about 200 aminoacid residues, and one or more longer XTEN carriers, wherein the VEGFbinding regions can bind and substantially sequester dimeric VEGF. Thereceptor binding fusion protein can also comprise a N-terminal XTEN,also illustrated in FIG. 6A-FIG. 6D, wherein the expression of thefusion protein in a host cell transformed with a suitable expressionvector comprising a polynucleotide encoding the fusion proteincomprising the N-terminal sequence is enhanced compared to theexpression of a corresponding fusion protein from a polynucleotide notcomprising the N-terminal XTEN encoding sequence. In the foregoingembodiments hereinabove described in this paragraph, the linkers as wellas the long carrier XTEN and the N-terminal XTEN can comprise a sequencethat can be a fragment of or that exhibits at least about 80% sequenceidentity, or 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to asequence selected from any one of Tables 4 or 11-15. In anotherembodiment, the linker can be a sequence in which at least 80% of theresidues are comprised of amino acids glycine, serine, and/or glutamate,such as, but not limited to a sequence with about 80-100% sequenceidentify to the sequence GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 1),or a portion or a multimer thereof.

In a particular feature of the above described embodiments, the cytokinebinding fusion proteins can be produced as functionally-active monomers,a characteristic which is believed to require fewer manufacturing stepsand result in a more homogenous product compared to constructs requiringa dimerization process (e.g., Fc conjugates) in order to recover afunctional molecule.

Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. A non-limiting example of a bispecificbinding fusion protein would be one with two targeting moieties that maybind to two different epitopes of the ErbB2 protein. For example, onetargeting moiety may bind an epitope in Domain 1 of ErbB2 such as the7C2/7F3 epitope, the other may bind a different ErbB2 epitope, e.g. the4D5 epitope. Other such bispecific binding fusion proteins may combinean ErbB2 binding site with binding site(s) for EGFR, ErbB3 and/or ErbB4.Alternatively, an anti-ErbB2 targeting moiety may be combined with atargeting moiety that binds to a triggering molecule on a leukocyte suchas a T-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG(FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as tofocus cellular defense mechanisms to the ErbB2-expressing cell.Bispecific binding fusion proteins can be prepared with targetingmoieties that are multivalent scFv, multivalent domain antibodies, ordiabodies.

(d) Configurations of Binding Fusion Proteins

The invention contemplates different configurations of binding fusionproteins, including but not limited to those comprising targetingmoieties characterized as scFv, diabodies, domain antibodies,bifunctional antibodies or cytokine binding fusion proteins, linked toone or more XTEN, and optionally having one or more linkers. For bindingfusion proteins with a single targeting moiety, the invention provides amonomeric binding fusion protein of formula I:(XTEN)_(x)-TM-(XTEN)_(y)  Iwherein independently for each occurrence: XTEN is an extendedrecombinant polypeptide as described above, x is either 0 or 1; y iseither 0 or 1, wherein x+y≥1; and TM is a targeting moiety with bindingaffinity to a target, preferably a target selected from Table 1 or Table2. In one embodiment, the TM comprises two or more binding domains thatmay be joined by a linker sequence of 1 to about 300 amino acid residueshaving a flexible, unstructured conformation. In one embodiment, thelinker sequence is an XTEN having at least about 12 to about 300 aminoacids exhibiting a flexible, unstructured characteristic, such as afragment of an XTEN, such that the binding domains can thereby mutuallyorientate relative to each other and the ligand and adopt a constrainedconfiguration to bind the target ligand. In another embodiment, thelinker can be a sequence in which at least 80% of the residues arecomprised of amino acids glycine, serine, and/or glutamate, such as, butnot limited to a sequence with about 80-100% sequence identify to thesequence GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 1), or a portion ora multimer thereof.

In another embodiment, wherein the TM comprises two or more bindingdomains, the binding domains may be identical or they may be different,and can comprise, for example, sequences derived from such V_(L) orV_(H) sequences, receptor binding domains, or Ig-like binding domains asnecessary to bind with sufficient affinity to the target ligand.

In another embodiment, the invention provides a multivalent bindingfusion protein with two binding moieties of formula II:(XTEN)_(x)-TM1-L-TM2-(XTEN)_(y)  IIwherein independently for each occurrence: XTEN is an extendedrecombinant polypeptide as described above, x is either 0 or 1; y iseither 0 or 1, wherein x+y≥1; TM1 is a targeting moiety with bindingaffinity to a target, preferably a target selected from Table 1 or Table2; TM2 is a targeting moiety with binding affinity to the target ligandthat may be identical or may be different to TM1; and L is a linkersequence having between 1 to about 300 amino acid residues wherein thelinker sequence is covalently bound to the C terminus of TM1 and the Nterminus of TM2. In one embodiment, the respective TM may each comprisetwo or more binding domains that may be joined by an additional linkersequence of 1 to about 300 amino acid residues having a flexible,unstructured conformation. In one embodiment, the linker sequence is anXTEN or a fragment of an XTEN from any one of Tables 4 or 11-15 havingat least about 12 to about 300 amino acids exhibiting a flexible,unstructured characteristic such that the binding domains can therebymutually orientate relative to each other and the ligand and adopt aconstrained configuration to bind to the target ligand. In anotherembodiment, the linker can be a sequence in which at least 80% of theresidues are comprised of amino acids glycine, serine, and/or glutamate,such as, but not limited to a sequence with about 80-100% sequenceidentify to the sequence GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 1),or a portion or a multimer thereof. In another embodiment, wherein theTM comprises two or more binding domains, the binding domains may beidentical or they may be different, and can comprise, for example,sequences derived from such V_(L) or V_(H) sequences, receptor bindingdomains, or Ig-like binding domains as necessary to bind with sufficientaffinity to the target ligand.

In another embodiment, the invention provides a multivalent bindingfusion protein with two binding moieties of formula III:TM1-XTEN-TM2  IIIwherein independently for each occurrence: XTEN is an extendedrecombinant polypeptide as described above; TM1 is a targeting moietywith binding affinity to a target, preferably a target selected fromTable 1 or Table 2; and TM2 is a targeting moiety with binding affinityto a target, preferably a target selected from Table 1 or Table 2 thatmay be identical or may be different to TM1. In one embodiment, therespective TM may each comprise two or more binding domains that may bejoined by an additional linker sequence of 1 to about 300 amino acidresidues having a flexible, unstructured conformation. In oneembodiment, the linker sequence is an XTEN or a fragment of an XTEN fromany one of Tables 4 or 11-15 having at least about 12 to about 300 aminoacids exhibiting a flexible, unstructured characteristic such that thebinding domains can thereby mutually orientate relative to each otherand the ligand and adopt a constrained configuration to bind to thetarget ligand. In another embodiment, the linker can be a sequence inwhich at least 80% of the residues are comprised of amino acids glycine,serine, and/or glutamate, such as, but not limited to a sequence withabout 80-100% sequence identify to the sequenceGSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 1), or a portion or amultimer thereof. In another embodiment, wherein the TM comprises two ormore binding domains, the binding domains may be identical or they maybe different, and can comprise, for example, sequences derived from suchV_(L) or V_(H) sequences, receptor binding domains, or Ig-like bindingdomains as necessary to bind with sufficient affinity to the targetligand.

In another embodiment, the invention provides a multivalent bindingfusion protein with three binding moieties of formula IV:(XTEN)_(x)-TM1-L1-TM2-L2-TM3-(XTEN)_(y)  IVwherein independently for each occurrence: XTEN is an extendedrecombinant polypeptide as described above, x is either 0 or 1; y iseither 0 or 1, wherein x+y≥1; TM1 is a targeting moiety with bindingaffinity to a target, preferably a target selected from Table 1 or Table2; TM2 is a targeting moiety with binding affinity to a target,preferably a target selected from Table 1 or Table 2 that may beidentical or may be different to TM1; TM3 is a targeting moiety withbinding affinity to a target, preferably a target selected from Table 1or Table 2 that may be identical or may be different to either TM1 orTM2; L1 is a linker sequence having between 1 to about 300 amino acidresidues as described for formula II, and wherein the linker sequence iscovalently bound to the C terminus of TM1 and the N terminus of TM2; andL2 is a linker sequence that may be identical to or different from L1,having between 1 to about 300 amino acid residues as described as forformula II, and wherein the linker sequence is covalently bound to the Cterminus of TM2 and the N terminus of TM3. In one embodiment, therespective TM may each comprise two or more binding domains that may bejoined by an additional linker sequence of 1 to about 300 amino acidresidues having a flexible, unstructured conformation. In oneembodiment, the linker sequence is an XTEN having at least about 12 toabout 300 amino acids exhibiting a flexible, unstructured characteristicsuch that the binding domains can thereby mutually orientate relative toeach other and the ligand and adopt a constrained configuration to bindto the target ligand. In another embodiment, wherein the TM comprisestwo or more binding domains, the binding domains may be identical orthey may be different, and can comprise, for example, sequences derivedfrom such V_(L) or V_(H) sequences, receptor binding domains, or Ig-likebinding domains as necessary to bind with sufficient affinity to thetarget ligand.

The invention contemplates additional and alternative configurations ofthe foregoing embodiments, including additional targeting moieties,binding regions, linkers and XTEN configured in alternative permutationsof order, N- to C-terminus, for the various components. The bindingfusion proteins of the embodiments disclosed herein exhibit one or moreor any combination of the properties and/or the embodiments as detailedherein.

(e) Configurations of Binding Fusion Proteins with Spacer and CleavageSequences

The invention contemplates configurations of binding fusion proteins,including but not limited to those comprising targeting moietiescharacterized as scFv, diabodies, domain antibodies, bifunctionalantibodies or cytokine binding fusion proteins, in which the XTEN may belinked to targeting moieties by spacer sequences incorporated into oradjacent to the XTEN that are designed to incorporate or enhance afunctionality or property to the composition, or as an aid in theassembly or manufacture of the binding fusion protein compositions. Suchproperties include, but are not limited to, inclusion of cleavagesequence(s) to permit release of components, inclusion of amino acidscompatible with nucleotide restrictions sites to permit linkage ofXTEN-encoding nucleotides to targeting moiety-encoding nucleotides orthat facilitate construction of expression vectors, or to reduce sterichindrance in regions of the fusion proteins.

A spacer sequence can be introduced between an XTEN sequence and atargeting moiety component to decrease steric hindrance such that thetargeting moiety component may assume its desired tertiary structureand/or interact appropriately with its target. For spacers and methodsof identifying desirable spacers, see, for example, George, et al.(2003) Protein Engineering 15:871-879, specifically incorporated byreference herein. In one embodiment, the spacer comprises one or morepeptide sequences that are between 1-50 amino acid residues in length,or about 1-25 residues, or about 1-10 residues in length. Spacersequences, exclusive of cleavage sites, can comprise any of the 20natural L amino acids, and will preferably have XTEN-like properties inthat 1) they will comprise hydrophilic amino acids that are stericallyunhindered such as, but not limited to, glycine (G), alanine (A), serine(S), threonine (T), glutamate (E), proline (P) and aspartate (D); and 2)will be substantially non-repetitive. In some cases, the spacer can bepolyglycines or polyalanines, or is predominately a mixture ofcombinations of glycine, serine and alanine residues. In one embodiment,a spacer sequence, exclusive of cleavage site amino acids, has about 1to 10 amino acids that consist of amino acids selected from glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), and proline (P)and are substantially devoid of secondary structure; e.g., less thanabout 10%, or less than about 5% as determined by the Chou-Fasman and/orGOR algorithms. In one embodiment, the spacer sequence is GPEGPS (SEQ IDNO: 145). In another embodiment, the spacer sequence is GPEGPS (SEQ IDNO: 145) linked to a cleavage sequence of Table 7. In addition, spacersequences are designed to avoid the introduction of T-cell epitopes;determination of which are described above and in the Examples.

In one embodiment, the binding fusion protein comprises one or morespacer sequences linked at the junction(s) between the payload sequenceand the one more XTEN incorporated into the fusion protein, wherein thespacer sequences comprise amino acids that are compatible withnucleotides encoding restriction sites. In another embodiment, thebinding fusion protein comprises one or more spacer sequences linked atthe junction(s) between the payload sequence and the one more XTENincorporated into the fusion protein wherein the spacer sequencescomprise amino acids that are compatible with nucleotides encodingrestriction sites and the amino acids and the one more spacer sequenceamino acids are chosen from glycine (G), alanine (A), serine (S),threonine (T), glutamate (E), and proline (P). In another embodiment,the binding fusion protein comprises one or more spacer sequences linkedat the junction(s) between the payload sequence and the one more XTENincorporated into the fusion protein wherein the spacer sequencescomprise amino acids that are compatible with nucleotides encodingrestriction sites and the one more spacer sequences are chosen from thesequences of Table 6. The exact sequence of each spacer sequence ischosen to be compatible with cloning sites in expression vectors thatare used for a particular binding fusion protein construct. Forembodiments in which a single XTEN is attached to the N- or C-terminus,only a single spacer sequence at the junction of the two componentswould be required. As would be apparent to one of ordinary skill in theart, the spacer sequences comprising amino acids compatible withrestriction sites could be omitted from the construct when an entirefusion protein gene is synthetically generated, rather than ligatedusing targeting moiety and XTEN encoding genes.

TABLE 6 Spacer Sequences Compatible with Restriction Sites SpacerSequence Restriction Enzyme GSPG (SEQ ID NO: 146) BsaI ETET (SEQ ID NO:147) BsaI PGSSS (SEQ ID NO: 148) BbsI GAP AscI GPA FseI GPSGP (SEQ IDNO: 149) SfiI AAA SacII TG AgeI GT KpnI

In some embodiments, a spacer sequence in a binding fusion proteincomposition comprises one or more cleavage sequences, which areidentical or different, wherein the cleavage sequence may be acted on bya protease to release the XTEN sequence(s) from the fusion protein. Inone embodiment, the incorporation of the cleavage sequence into thebinding fusion protein is designed to permit release of an targetingmoiety that becomes active or more active upon its release from the XTENcomponent. The cleavage sequences are located sufficiently close to thetargeting moiety sequences, generally within 18, or within 12, or within6, or within 2 amino acids of the targeting moiety sequence, such thatany remaining residues attached to the targeting moiety after cleavagedo not appreciably interfere with the activity (e.g., such as binding toa target), yet provide sufficient access to the protease to be able toeffect cleavage of the cleavage sequence. In some cases, the bindingfusion protein comprising the cleavage sequences will also have one ormore spacer sequence amino acids between the targeting moiety and thecleavage sequence or the XTEN and the cleavage sequence to facilitateaccess of the protease to the cleavage sequence; the spacer amino acidscomprising any natural amino acid, including glycine, serine and alanineas preferred amino acids. In one embodiment, the cleavage site is asequence that can be cleaved by a protease endogenous to the mammaliansubject such that the fusion protein can be cleaved after administrationto a subject. In one embodiment of the foregoing construct, thetargeting moiety that is released from the fusion protein by cleavage ofthe cleavage sequence exhibits at least about a two-fold, or at leastabout a three-fold, or at least about a four-fold, or at least about afive-fold, or at least about a six-fold, or at least about a eight-fold,or at least about a ten-fold, or at least about a 20-fold increase inactivity compared to the intact binding fusion protein.

Examples of cleavage sites contemplated by the invention include, butare not limited to, a polypeptide sequence cleavable by a mammalianendogenous protease selected from FXIa, FXIIa, kallikrein, FVIIIa,FVIIIa, FXa, FIIa (thrombin), Elastase-2, granzyme B, MMP-12, MMP-13,MMP-17 or MMP-20, or by non-mammalian proteases such as TEV,enterokinase, PreScission™ protease (rhinovirus 3C protease), andsortase A. Sequences known to be cleaved by the foregoing proteases andothers are known in the art. Exemplary cleavage sequences contemplatedby the invention and the respective cut sites within the sequences arepresented in Table 7, as well as sequence variants thereof.

In one embodiment, the invention provides binding fusion proteinscomprising one or more cleavage sequences operably positioned to releasethe targeting moiety from the fusion protein upon cleavage, wherein theone or more cleavage sequences has at least about 86%, or at least about92% or greater sequence identity to a sequence selected from Table 7.

In some embodiments, only the two or three amino acids flanking bothsides of the cut site (four to six amino acids total) are incorporatedinto the cleavage sequence that, in turn, is incorporated into thefusion proteins of the embodiments. In other embodiments, theincorporated cleavage sequence of Table 7 can have one or more deletionsor insertions or one or two or three amino acid substitutions for anyone or two or three amino acids in the known sequence, wherein thedeletions, insertions or substitutions result in reduced or enhancedsusceptibility but not an absence of susceptibility to the protease,resulting in an ability to tailor the rate of release of the targetingmoiety from the XTEN. Exemplary substitutions are shown in Table 7.

TABLE 7 Protease Cleava2e Sequences Exemplary Protease Acting CleavageSEQ ID SEQ ID Upon Sequence Sequence NO: Minimal Cut Site* NO: FXIaKLTR↓AET 150 KD/FL/T/R↓VA/VE/GT/GV FXIa DFTR↓VVG 151KD/FL/T/R↓VA/VE/GT/GV FXIIa TMTR↓IVGG 152 NA Kallikrein SPFR↓STGG 153-/-/FL/RY↓SR/RT/-/- FVIIa LQVR↓IVGG 154 NA FIXa PLGR↓IVGG 155-/-/G/R↓-/-/-/- FXa IEGR↓TVGG 156 IA/E/GFP/R↓STI/VFS/-/G FIIa (thrombin)LTPR↓SLLV 157 -/-/PLA/R↓SAG/-/-/- Elastase-2 LGPV↓SGVP 158-/-/-/VIAT↓-/-/-/- Granzyme-B VAGD↓SLEE 159 V/-/-/D↓-/-/-/- MMP-12GPAG↓LGGA 160 G/PA/-/G↓L/-/G/- 161 MMP-13 GPAG↓LRGA 162 G/P/-/G↓L/-/GA/-163 MMP-17 APLG↓LRLR 164 -/PS/-/-↓LQ/-/LT/- MMP-20 PALP↓LVAQ 165 NA TEVENLYFQ↓G 166 ENLYFQG↓S 167 Enterokinase DDDK↓IVGG 168 DDDK↓IVGG 169Protease 3C LEVLFQ↓GP 170 LEVLFQ↓GP 171 (PreScission ™) Sortase ALPKT↓GSES 172 L/P/KEAD/T↓G/-/EKS/S 173 ↓indicates cleavage site NA: notapplicable *the listing of multiple amino acids before, between, orafter a slash indicate alternative amino acids that can be substitutedat the position; ″-″ indicates that any amino acid may be substitutedfor the corresponding amino acid indicated in the middle column

(f) Methods of Use of Binding Fusion Proteins

In another aspect, the invention provides a method of for achieving abeneficial effect in a disease, disorder or condition mediated by abinding fusion protein. In one embodiment, the invention provides theuse of a binding fusion protein derived from a parental antibody thatbinds to a target selected from the group consisting of the targets ofTable 1 in treatment of a disease, disorder or condition to a subject inneed thereof by the administration of a therapeutically effective amountof the binding fusion protein, wherein said administration leads to theeradication or amelioration of one or more of the physiological orclinical symptoms associated with the underlying disorder such that animprovement is observed in the subject, notwithstanding that the subjectmay still be afflicted with the underlying disorder. In anotherembodiment, the invention provides a method of treating a disease,disorder, or condition in a mammal comprising administering to themammal a therapeutically effective amount of a binding fusion proteincomprising one or more targeting moieties directed to one or moretargets selected from Table 1, linked to one or more XTEN sequencesmolecules and, optionally, one or more linkers, to form the bindingfusion protein, wherein the linkage does not substantially alter theessential functional properties of the binding fusion protein of bindingaffinity and sustained terminal half-life or reduced serum clearancerate as compared to that of the parental targeting moiety from which thebinding fusion protein is derived, and wherein the administration of thebinding fusion protein achieves a beneficial therapeutic effect. Theeffective amount can produce a beneficial effect in helping to treat(e.g., cure or reduce the severity) or prevent (e.g., reduce thelikelihood of onset or severity) a disease, disorder or condition, suchas, but not limited to a cancer, a cardiovascular disease or condition,an infectious disease, an inflammatory condition, a respiratorycondition, organ transplant rejection, or a metabolic disease mediatedby or associated with one or more targets, preferably selected fromTable 1.

In one embodiment, the method comprises administering atherapeutically-effective amount of a pharmaceutical compositioncomprising a binding fusion protein composition comprising one or moretargeting moieties linked to one or more XTEN sequence(s) and at leastone pharmaceutically acceptable carrier to a subject in need thereofthat results in an improvement in at least one parameter, physiologiccondition, or clinical outcome mediated by the targeting moietycomponent(s). The method contemplates administration of thepharmaceutical composition by any route appropriate for the disease,disorder or condition being treated, including subcutaneously,intramuscularly, intravitreally, or intravenously.

The methods of the invention include administration of consecutive dosesof a therapeutically effective amount of the pharmaceutical compositionfor a period of time sufficient to achieve and/or maintain the desiredparameter or clinical effect, and such consecutive doses of atherapeutically effective amount establishes the therapeuticallyeffective dose regimen for the pharmaceutical composition; i.e., theschedule for consecutively administered doses, wherein the doses aregiven in therapeutically effective amounts to result in a sustainedbeneficial effect on any clinical sign or symptom, aspect, measuredparameter or characteristic of a metabolic disease state or condition,including, but not limited to, those described herein.

A therapeutically effective amount of the pharmaceutical composition mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the binding fusion protein are outweighed by thetherapeutically beneficial effects. A prophylactically effective amountrefers to an amount of pharmaceutical composition required for theperiod of time necessary to achieve the desired prophylactic result.

For the inventive methods, longer acting binding fusion proteincompositions or pharmaceutical compositions are preferred, so as toimprove patient convenience, to increase the interval between doses andto reduce the amount of drug required to achieve a sustained effect. Inone embodiment, a method of treatment comprises administration of atherapeutically effective dose of a binding fusion protein to a subjectin need thereof that results in a gain in time spent within atherapeutic window established for the fusion protein of thepharmaceutical composition compared to the corresponding targetingmoiety component(s) not linked to the XTEN and administered at acomparable dose to a subject. In one embodiment, the gain in time spentwithin the therapeutic window is at least about three-fold, or at leastabout four-fold, or at least about five-fold, or at least aboutsix-fold, or at least about eight-fold, or at least about 10-fold, or atleast about 20-fold, or at least about 40-fold compared to thecorresponding targeting moiety component not linked to the XTEN andadministered at a comparable dose to a subject. The methods furtherprovide that administration of multiple consecutive doses of apharmaceutical composition administered using a therapeuticallyeffective dose regimen to a subject in need thereof can result in a gainin time between consecutive C_(max) peaks and/or C_(min) troughs forblood levels of the binding fusion protein compared to the correspondingtargeting moieties not linked to the XTEN. In the foregoing embodiment,the gain in time spent between consecutive C_(max) peaks and/or C_(min)troughs can be at least about three-fold, or at least about four-fold,or at least about five-fold, or at least about six-fold, or at leastabout eight-fold, or at least about 10-fold, or at least about 20-fold,or at least about 40-fold compared to the corresponding targeting moietycomponent(s) not linked to the XTEN and administered using a comparabledose regimen established for that targeting moiety. In the embodimentshereinabove described in this paragraph the administration of the fusionprotein or pharmaceutical composition can result in an improvement in atleast one parameter known to be useful for assessing the subjectdiseases, conditions or disorders) using a lower unit dose in moles offusion protein compared to the corresponding targeting moietycomponent(s) not linked to the XTEN and administered at a comparableunit dose or dose regimen to a subject.

In one embodiment, the administration of a binding fusion protein orpharmaceutical composition can result in an improvement in one of theclinical, biochemical or physiologic parameters that is greater thanthat achieved by administration of the targeting moiety component notlinked to XTEN, determined using the same assay or based on a measuredclinical parameter. In another embodiment, administration of the bindingfusion protein or pharmaceutical composition can result in improvementtwo or more clinical or metabolic-related parameters, each mediated byone of the different targeting moieties that collectively result in anenhanced effect compared the targeting moiety component not linked tothe XTEN, determined using the same assays or based on measured clinicalparameters. In another embodiment, administration of the binding fusionprotein or pharmaceutical composition can result in activity in one ormore of the clinical or biochemical or physiologic parameters that is oflonger duration than the activity of one of the single targeting moietycomponents not linked to the XTEN, determined using that same assay orbased on a measured clinical parameter.

In one embodiment, the binding fusion protein is used to treatVEGF-mediated disorders. In particular, the invention provides a methodfor treating a VEGF-mediated disease in a human patient with a bindingfusion protein comprising one or more of the targeting moieties thatbinds to human VEGF, wherein the binding reduces the ability of the VEGFto bind its cognate receptor. Such binding fusion proteins can haveprophylactic and therapeutic applications in a broad spectrum ofVEGF-mediated disorders, including pathologies supported by blood vesselproliferation, i.e. angiogenesis, in a manner similar to the applicationof anti-VEGF antibodies in the treatment of such disease indicationsthat is known in the art, which treatment indications include solidtumors ((Kim et al. Nature 362:841-844 (1993); Warren et al. J. Clin.Invest. 95:1789-1797 (1995); Borgstrom et al. Cancer Res. 56:4032-4039(1996); and Melnyk et al. Cancer Res. 56:921-924 (1996)) and intraocularneovascular syndromes such as proliferative retinopathies andage-related macular degeneration (AMD) (Adamis et al. Arch. Ophthalmol.114:66-71 (1996)).

Fusion proteins comprising the XTEN of the invention can approximate thein vivo pharmacokinetics (e.g. terminal half-life) of full-lengthantibody. Given these characteristics, it is believed that the bindingfusion proteins of the invention comprising anti-VEGF targeting moietiesdisplay the same or substantially similar in vivo activities as fulllength anti-VEGF monoclonal antibody across a range of differentparameters, including pharmacokinetic characteristics and therapeuticendpoints in an animal tumor model, supporting the utility of thebinding fusion proteins in the same broad spectrum of neovasculardisease indications that responds to full length anti-VEGF antibodytreatment.

Any binding fusion protein disclosed herein that comprises a targetingmoiety derived from an anti-VEGF antibody or fragment can beadvantageously utilized in a method of treating a VEGF-mediated diseaseor disorder, such as neovascular disorders. In one embodiment, theinvention provides a method of treating a neovascular disorder in ahuman patient comprising administering to the patient a therapeuticallyeffective amount of a binding fusion protein or pharmaceuticalcomposition wherein at least one targeting moiety in the binding fusionprotein comprises an antigen binding site that binds to human VEGF.

In another embodiment, the invention provides a method of treating asolid tumor disorder in a human patient comprising administering to thepatient an effective amount of a binding fusion protein orpharmaceutical composition wherein at least one targeting moiety in thebinding fusion protein comprises an antigen binding site that binds tohuman VEGF. In yet another embodiment, the solid tumor disorder in theforegoing method is selected from the group consisting of breastcarcinomas, lung carcinomas, gastric carcinomas, esophageal carcinomas,colorectal carcinomas, liver carcinomas, ovarian carcinomas, thecomas,arrhenoblastomas, cervical carcinomas, endometrial carcinoma,endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma,head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas,hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma,cavernous hemangioma, hemangioblastoma, pancreas carcinomas,retinoblastoma, astrocytoma, glioblastoma, Schwannoma,oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma,osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroidcarcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma,abnormal vascular proliferation associated with phakomatoses, edema(such as that associated with brain tumors), and Meigs' syndrome.

In still another embodiment, the invention provides a method of treatingan intraocular neovascular disorder in a human patient comprisingadministering to the patient a therapeutically effective amount of abinding fusion protein or pharmaceutical composition wherein at leastone targeting moiety comprises an antigen binding site that binds tohuman VEGF. In a further embodiment, the intraocular neovasculardisorder is selected from the group consisting of diabetic and otherproliferative retinopathies including retinopathy of prematurity,retrolental fibroplasia, neovascular glaucoma, and age-related maculardegeneration.

In another embodiment, the invention provides a method of inhibitingangiogenesis in a human patient comprising administering to the patientan effective amount of a binding fusion protein wherein at least onetargeting moiety in the composition comprises an antigen binding sitethat binds to human VEGF.

In another embodiment, the binding fusion protein is used to treatdisorders mediated by HER2-expressing cells. The invention provides amethod for treating a human disease mediated by HER2-expressing cellswith a binding fusion protein composition that is derived from aparental antibody that binds to HER2. Such compositions haveprophylactic and therapeutic applications in a broad spectrum ofHER2-expressing cell-mediated disorders, including pathologies supportedby the proliferation of cells expressing HER2, such as cancerscharacterized by over-expression of HER2, in a manner similar to theapplication of full length anti-Her2 antibodies in the treatment of suchdisease indications that is known in the art, which treatmentindications include HER2-overexpressing breast, ovarian and lungcancers. The choice of a targeting moiety for a binding fusion proteinto be used in the method can be determined by in vitro binding assays orin vitro cell-killing assays as described in the Examples or are knownin the art. For example, a candidate binding fusion protein against HER2can be used in cytotoxicity tests using cell cultures of human breastcancer lines such as MCF-7, CAMA-1, SKBR-3, and BT-20, such as describedin U.S. Pat. No. 4,753,894. In one embodiment, the invention provides amethod of treating a HER2-expressing cell mediated disorder in a humanpatient comprising administering to the patient a therapeuticallyeffective amount of a binding fusion protein or pharmaceuticalcomposition wherein at least one targeting moiety in the binding fusionprotein comprises an antigen binding site that binds to HER2. Thedisorder can be a HER2-expressing cell proliferative disorder, includinga benign or malignant tumor characterized by the over-expression of theErbB2 receptor, e.g. a cancer, such as, breast cancer, squamous cellcancer, small-cell lung cancer, non-small cell lung cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, coloncancer, colorectal cancer, endometrial carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer. In addition, the invention contemplates the use of the foregoingcomposition in place of full-length anti-Her2 antibody in the treatmentof HER2-overexpressing cancers as described in U.S. Pat. No. 5,725,856.

In another embodiment, the invention provides a method of treatingdisorders mediated by CD20-Expressing cells. The invention provides amethod for treating a human disease mediated by CD20-expressing cellswith a binding fusion protein composition that is derived from aparental antibody that binds to human CD20. Such compositions haveprophylactic and therapeutic applications in a broad spectrum ofCD20-expressing cell-mediated disorders, including pathologies supportedby the proliferation of CD20-expressing cells, such as cancers ofCD20-expressing cells, in a manner similar to the application of fulllength anti-CD20 antibodies in the treatment of such disease indicationsknown in the art, which treatment indications include B-lymphocyticlymphomas, as described in U.S. Pat. No. 6,682,734.

In another embodiment, the invention provides a method of treatingdisorders mediated by CD18-expressing cells. The invention provides amethod for treating a human disease mediated by CD18-expressing cellswith a binding fusion protein composition that is derived from aparental antibody that binds to human CD18. Such compositions haveprophylactic and therapeutic applications in a broad spectrum ofCD18-expressing cell-mediated disorders, including pathologies supportedby leukocyte adhesion, in a manner similar to the application of fulllength anti-CD18 antibodies in the treatment of such disease indicationsknown in the art, which treatment indications include acute myocardialinfarction and stroke. In one embodiment, the invention provides amethod of treating a disorder in a human patient mediated by aCD18-expressing cell, comprising administering to the patient atherapeutically effective amount of a binding fusion protein wherein atleast one targeting moiety in the composition comprises an antigenbinding site that binds to human CD18. In another embodiment, theCD18-expressing cell-mediated disorder is an inflammatory disorder, suchas an ischemic reperfusion disorder, including acute myocardialinfarction and stroke. In addition, the invention contemplates the useof the foregoing binding fusion protein in place of full-lengthanti-CD18 antibody in the treatment of stroke as described in PCTPublication WO 97/26912.

In another embodiment, the invention provides a method of treating aLFA-1-mediated disorder in a human, comprising administering to thepatient a therapeutically effective amount of a binding fusion proteinwherein at least one targeting moiety in the composition comprises atargeting moiety that binds to human CD18. In addition, the inventioncontemplates the use of the foregoing binding fusion protein in place offull-length anti-CD18 antibody in the treatment of a LFA-1-mediateddisorder, such as psoriasis and graft rejection, in a human patient asdescribed in U.S. Pat. No. 5,622,700.

In another embodiment, the invention provides a method of treatingdisorders mediated by CD11a-expressing cells. In one embodiment, theinvention provides a method for treating a human disease mediated by aCD11a-expressing cell with a binding fusion protein composition that isderived from a parental antibody that binds to human CD11a. Suchcompositions have prophylactic and therapeutic applications in a broadspectrum of CD11a-expressing cell-mediated disorders, includingpathologies supported by leukocyte adhesion, in a manner similar to theapplication of full length anti-CD11a antibodies in the treatment ofsuch disease indications known in the art, which treatment indicationsinclude psoriasis, asthma, graft rejection, and multiple sclerosis. Inanother embodiment, the invention provides a method of treating aLFA-1-mediated disorder in a human, comprising administering to thepatient a therapeutically effective amount of a binding fusion proteinwherein at least one targeting moiety in the composition comprises anantigen binding site that binds to human CD11a. In addition, theinvention contemplates the use of the foregoing binding fusion proteinin place of full-length anti-CD11a antibody in the treatment of aLFA-1-mediated disorder, such as psoriasis and graft rejection, in ahuman patient as described in U.S. Pat. No. 5,622,700. In anotheraspect, the invention contemplates the use of the foregoing bindingfusion proteins in place of full-length anti-CD11a antibody in thetreatment of LFA-1-mediated disorders in a human patient as described inU.S. Pat. No. 6,037,454.

In another embodiment, the invention provides a method of treatingIgE-mediated disorders. In one embodiment, the invention provides amethod for treating an IgE-mediated disorder in a human patient with abinding fusion protein composition that is derived from a parentalantibody that binds to human IgE. Such compositions have prophylacticand therapeutic applications in a broad spectrum of IgE-mediateddisorders, including pathologies characterized by the overproductionand/or hypersensitivity to the immunoglobulin IgE, in a manner similarto the application of anti-IgE antibodies in the treatment of suchdisease indications known in the art, which treatment indicationsinclude allergic diseases, such as allergic asthma and allergicrhinitis. In one embodiment, the invention provides a method of treatingan IgE-mediated disorder in a human patient comprising administering tothe patient a therapeutically effective amount of a binding fusionprotein described wherein at least one targeting moiety comprises anantigen binding site that binds to human IgE. In another embodiment, theIgE-mediated disorder is an allergic disease. In yet another embodiment,the IgE-mediated disorder is allergic asthma. In still anotherembodiment, the IgE-mediated disorder is allergic rhinitis.

In a further embodiment, the invention provides a method of treating anIgE-mediated disorder in a human patient comprising administering to thepatient a therapeutically effective amount of a binding fusion proteinwherein at least one targeting moiety in the composition comprises anantigen binding site that competes with human Fc epsilonRI for bindingto human IgE. In yet another embodiment, the invention provides a methodof treating an IgE-mediated disorder in a human patient comprisingadministering to the patient a therapeutically effective amount of abinding fusion protein wherein at least one antibody fragment in thebinding fusion protein comprises an antigen binding site that binds tomembrane-bound IgE on the surface of human B-lymphocytes but does notbind to soluble IgE bound to Fc epsilon RI receptor on the surface ofhuman basophils. In addition, the invention contemplates the use of anyof the foregoing binding fusion proteins in place of full lengthanti-human IgE antibody in the treatment of an IgE-mediated disorder,such as allergic diseases including allergic asthma and allergicrhinitis, in a human patient as described in PCT Application No. WO99/01556. In another aspect, the invention contemplates the use of anyof the foregoing binding fusion proteins in place of full lengthanti-human IgE antibody in the treatment of allergic asthma in a humanpatient as described in WO 97/04807.

In another embodiment, the invention provides a method of treating anallergic disease in a human patient comprising administering to thepatient a therapeutically effective amount of a binding fusion proteinwherein at least one targeting moiety in the binding fusion proteincomprises an antigen binding site that competes with human Fc epsilon RIfor binding to human IgE. In yet another embodiment, the inventionprovides a method of treating an allergic disease in a human patientcomprising administering to the patient a therapeutically effectiveamount of a binding fusion protein wherein at least one targeting moietyin the binding fusion protein comprises an antigen binding site thatbinds to membrane-bound IgE on the surface of human B-lymphocytes butdoes not bind to soluble IgE bound to Fc epsilon RI receptor on thesurface of human basophils.

In another embodiment, the invention provides a method of treatingallergic asthma in a human patient comprising administering to thepatient a therapeutically effective amount of a binding fusion proteinwherein at least one targeting moiety in the binding fusion proteincomprises an antigen binding site that competes with human Fc epsilon RIfor binding to human IgE. In yet another embodiment, the inventionprovides a method of treating allergic asthma in a human patientcomprising administering to the patient a therapeutically effectiveamount of any binding fusion protein described in this Section (II)wherein at least one targeting moiety in the binding fusion proteincomprises an antigen binding site that binds to membrane-bound IgE onthe surface of human B-lymphocytes but does not bind to soluble IgEbound to Fc epsilonRI receptor on the surface of human basophils.

TNF-α-Mediated Disorders

In one embodiment, the invention provides a method for treating aTNF-α-mediated disease with a binding fusion protein that is derivedfrom a parental antibody that binds to human TNF-α. Such binding fusionproteins can have prophylactic and therapeutic applications in a broadspectrum of TNF-α-mediated disorders, including inflammatory disordersand immune disorders, in a manner similar to the application offull-length anti-human TNF-α antibodies in the treatment of such diseaseindications such as Crohn's disease, inflammatory bowel disease, andrheumatoid arthritis.

In one embodiment, the invention provides a method of treating aninflammatory disorder in a human patient comprising administering to thepatient a therapeutically effective amount of a binding fusion proteinwherein at least one targeting moiety in the binding fusion proteincomprises an antigen binding site that binds to human TNF-α. In anotherembodiment, the inflammatory disorder is Crohn's disease. In yet anotherembodiment, the inflammatory disorder is inflammatory bowel disease. Instill another embodiment, the inflammatory disorder is rheumatoidarthritis. The use of antibodies that bind to human TNF-α in thetreatment of inflammatory conditions have been described, for example,in U.S. Pat. Nos. 5,672,347, 5,656,272, and 5,698,195.

Tissue Factor-Mediated Disorders

In one embodiment, the invention provides a method for treating a tissuefactor-mediated disease with a binding fusion protein derived from aparental antibody that binds to human tissue factor. Such binding fusionproteins can have prophylactic and therapeutic applications in a broadspectrum of tissue factor-mediated disorders, including pathologiessupported by blood coagulation and in the treatment of such diseaseindications as deep vein thrombosis, arterial thrombosis,atherosclerosis, vascular stenosis, myocardial ischemic diseasesincluding acute myocardial infarction, reocclusion following angioplastyor atherectomy or thrombolytic treatment for acute myocardialinfarction, angina, cerebral ischemic diseases including stroke, venousthrombophlebitis, and pulmonary embolism. In one embodiment, theinvention provides a method of treating a tissue factor-mediated diseaseor disorder (such as the foregoing) in a human patient comprisingadministering to the patient a therapeutically effective amount of abinding fusion protein wherein at least one targeting moiety in thebinding fusion protein comprises an antigen binding site that binds tohuman tissue factor.

In another embodiment, the invention provides a method of inhibitingblood coagulation in a human patient comprising administering to thepatient a therapeutically effective amount of a binding fusion proteinwherein at least one targeting moiety in the binding fusion proteincomprises an antigen binding site that binds to human tissue factor,preventing the binding of coagulation factor VII.

Disorders Mediated by EGFR-Expressing Cells

In one embodiment, the invention provides a method for treating a humandisease mediated by EGFR-expressing cells with a of the binding fusionprotein that is derived from a parental antibody that binds to humanEGFR (a.k.a., ErbB-1 or Her1). Such binding fusion proteins can haveprophylactic and therapeutic applications in a broad spectrum ofEGFR-expressing cell-mediated disorders, including pathologies supportedby the proliferation of cells expressing EGFR, such as cancerscharacterized by over-expression of EGFR, including cancers of thebreast, ovary, head and neck, brain, bladder, pancreas, and lung.

In one embodiment, the invention provides a method of treating a cellproliferation disorder in a human patient characterized byover-expression of EGFR comprising administering to the patient atherapeutically effective amount of a binding fusion protein wherein atleast one targeting moiety in the binding fusion protein comprises anantigen binding site that binds to human EGFR. The disorder can be abenign or malignant tumor characterized by the over-expression of theEGFR, e.g. a cancer, such as, breast cancer, squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, colon cancer, colorectalcancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer,liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer.

Disorders Mediated by CD3-Expressing Cells

In one embodiment, the invention provides a method for treating a humandisease or disorder mediated by CD3-expressing cells with a bindingfusion protein that is derived from a parental antibody that binds tohuman CD3. Such binding fusion proteins can have prophylactic andtherapeutic applications in a broad spectrum of CD3-expressingcell-mediated disorders, including conditions associated with theproliferation or activation of cells expressing CD3, such as immunedisorders mediated by T-lymphocytes and graft rejection in transplantrecipients. The use of anti-CD3 antibodies to treat diseases anddisorders has been described, for example, in U.S. Pat. No. 4,515,893.In another aspect, the invention contemplates the use of the foregoingbinding fusion protein in place of full length anti-human CD3 antibodyin the treatment of acute allograft rejection in kidney transplantrecipients as described for ORTHOCLONE OKT3 muromonab-CD3 in Physician'sDesk Reference, 52^(nd) Edition (1998), pp. 1971-1974.

Disorders Mediated by TAC-Expressing Cells

In one embodiment, the invention provides a method for treating a humandisease mediated by interleukin-2 receptor α-chain (TAC)-expressingcells with a binding fusion protein that is derived from a parentalantibody that binds to human TAC. Such binding fusion proteins can haveprophylactic and therapeutic applications in a broad spectrum ofTAC-expressing cell-mediated disorders, including conditions created bythe proliferation or activation of cells expressing TAC and immunedisorders mediated by T-lymphocytes or B-lymphocytes, including graftrejection in transplant recipients.

In one embodiment, the invention provides a method of treating adisorder in a human patient mediated by a TAC-expressing cell,comprising administering to the patient a therapeutically effectiveamount of a binding fusion protein wherein at least one targeting moietyin the binding fusion protein comprises an antigen binding site thatbinds to human TAC. In another embodiment, the TAC-expressingcell-mediated disorder is characterized by the activation orproliferation of T-lymphocytes or B-lymphocytes, including immunedisorders such as graft rejection in transplant recipients,graft-versus-host disease (GHVD), graft rejection in transplantrecipients, such as acute graft rejection in renal transplantrecipients, and autoimmune diseases such as Type I diabetes, multiplesclerosis, rheumatoid arthritis, systemic lupus erythematosus, andmyasthenia gravis. The use of antibodies to treat disorders mediated byinterleukin-2 receptor α-chain with antibodies has been described inU.S. Pat. No. 5,693,761.

(g) Binding Fusion Protein-Drug Compositions and XTEN-Drug Compositions

The present invention relates in part to compositions of binding fusionproteins covalently linked to a drug, resulting in a binding fusionprotein drug conjugate (“BFP-D”). In another aspect, the inventionrelates to compositions of XTEN covalently linked to a drug, resultingin an XTEN-drug conjugate (“XTEN-D”). In particular, the inventionprovides isolated BFP-D and XTEN-D compositions useful in the treatmentof diseases, disorders or conditions. In one embodiment, the BFP-Dcomprises one or more targeting moieties of the binding fusion proteindirected to an antigen, ligand, or receptor implicated in, associatedwith, or that modulates a disease, disorder or condition, while one ormore XTEN of the binding fusion protein can be designed to serve as acarrier to which the drug is conjugated, conferring a desired half-lifeor enhanced pharmaceutical property on the binding fusion protein, asdescribed more fully below, and the covalently-linked drug can beselectively delivered to a cell, tissue, or organ to effect apharmacologic, cytotoxic, or cytostatic effect. Thus, the BFP-Dgenerally comprises one or more of the following components: 1) XTEN; 2)targeting moiety; 3) cross-linker; and 4) drug. The XTEN-D generallycomprise one or more of the following components: 1) XTEN; 2)cross-linker; and 3) drug.

Exemplary embodiments of targeting moieties, XTEN and fusion proteins oftargeting moieties and XTEN have been described, above. The inventionprovides XTEN that further serve as a platform to which drugs can beconjugated, such that they serve as a “carrier”, conferring certaindesirable pharmacokinetic, chemical and pharmaceutical properties to thecompositions, amongst other properties described below.

In some embodiments, the XTEN component are engineered to incorporate adefined number of amino acid residues that contain reactive groups thatcan be used to conjugate to drugs and/or with cross-linking agents. Inone embodiment, the reactive amino acid is cysteine(“cysteine-engineered XTEN”). In another embodiment, the reactive aminoacid is lysine, which contains a positively charged hydrophilic ε-aminogroup (“lysine-engineered XTEN”). As used herein, a “cysteine-engineeredXTEN” means an XTEN protein, as defined above, further comprising about1 to about 100 cysteine; amino acids, or from 1 to about 50 cysteineamino acids, or from 1 to about 40 cysteine amino acids, or from 1 toabout 20 cysteine amino acids, or from 1 to about 10 cysteine aminoacids, or from 1 to about 5 cysteine amino acids that are available forconjugation to drug molecules. As used herein, a “lysine-engineeredXTEN” means an XTEN protein, as defined above, further comprising about1 to about 100 lysine amino acids, or from 1 to about 50 lysine aminoacids, or from 1 to about 40 lysine engineered amino acids, or from 1 toabout 20 lysine engineered amino acids, or from 1 to about 10 lysineengineered amino acids, or from 1 to about 5 lysine engineered aminoacids that are available for conjugation to drug molecules.

Generally, XTEN cysteine thiol groups are more reactive, i.e., morenucleophilic, towards electrophilic conjugation reagents than amine orhydroxyl groups. Cysteine residues have been introduced into proteins bygenetic engineering techniques to form covalent attachments to ligandsor to form new intramolecular disulfide bonds (Better et al (1994) J.Biol. Chem. 13:9644-9650; Bernhard et al (1994) Bioconjugate Chem.5:126-132; Greenwood et al (1994) Therapeutic Immunology 1:247-255; Tuet al (1999) Proc. Natl. Acad. Sci USA 96:4862-4867; Kanno et al (2000)J. of Biotechnology, 76:207-214; Chmura et al (2001) Proc. Nat. Acad.Sci. USA 98(15):8480-8484; U.S. Pat. No. 6,248,564).

In one embodiment, the invention provides an isolated compositioncomprising a cysteine-engineered XTEN conjugated by a cross-linker toone or more drug molecules, wherein the drug is selected from Table 9.In another embodiment, the invention provides an isolated compositioncomprising a targeted cysteine-engineered XTEN conjugated by across-linker to one or more drug molecules, wherein the drug is selectedfrom Table 9 and the targeted cysteine-engineered XTEN comprises one ormore targeting moieties that exhibit binding affinity to one or moretargets selected from Table 1 or Table 2. In another embodiment, theinvention provides an isolated composition comprising alysine-engineered XTEN conjugated by a cross-linker to one or more drugmolecules, wherein the drug is selected from Table 9. In anotherembodiment, the invention provides an isolated composition comprising atargeted lysine-engineered XTEN conjugated by a cross-linker to one ormore drug molecules, wherein the drug is selected from Table 9 and thetargeted cysteine-engineered XTEN comprises one or more targetingmoieties that exhibits binding affinity to one or more targets selectedfrom Table 1 or Table 2. In one embodiment of the foregoing, only asingle drug compound would be conjugated to the XTEN. In anotherembodiment, more than one drug compound may be conjugated to the XTEN byselective application of the conjugation methods and reactants, usingthe methods described herein or those known in the art.

In some cases, the compositions of the invention includecysteine-engineered XTEN where nucleotides encoding one or more aminoacids of an XTEN are replaced with a cysteine amino acid to create thecysteine-engineered XTEN gene. In other cases, oligonucleotides encodingone or more motifs of about 9 to about 14 amino acids comprising codonsencoding one or more cysteines are linked in frame with other oligosencoding XTEN motifs or full-length XTEN to create thecysteine-engineered XTEN gene. In one embodiment of the foregoing, wherethe one or more cysteines are inserted into an XTEN sequence during thecreation of the XTEN gene, nucleotides encoding cysteine can be linkedto codons encoding amino acids used in XTEN to create a cysteine-XTENmotif with the cysteine(s) at a defined position using the methodsdescribed herein (see Example 61 and FIGS. 40-41), or by standardmolecular biology techniques, and the motifs subsequently assembled intothe gene encoding the full-length cysteine-engineered XTEN. In suchcases, where, for example, nucleotides encoding a single cysteine areadded to the DNA encoding a motif selected from Table 3, the resultingmotif would have 13 amino acids, while incorporating two cysteines wouldresult in a motif having 14 amino acids, etc. In other cases, acysteine-motif can be created de novo and be of a pre-defined length andnumber of cysteine amino acids by linking nucleotides encoding cysteineto nucleotides encoding one or more amino acid residues used in XTEN(e.g., G, S, T, E, P, A) at a defined position, and the encoding motifssubsequently assembled by annealing with other XTEN-encoding motifsequences into the gene encoding the full-length XTEN, as describedherein and illustrated in FIGS. 7-8. In cases where a lysine-engineeredXTEN is utilized to make the compositions of the invention, theapproaches described above would be performed with codons encodinglysine instead of cysteine. Thus, by the foregoing, a new XTEN motif canbe created that could comprise about 9-14 amino acid residues and haveone or more reactive amino acids; i.e., cysteine or lysine. Non-limitingexamples of motifs suitable for use in an engineered XTEN that contain asingle cysteine or lysine are:

(SEQ ID NO: 174) GGSPAGSCTSP (SEQ ID NO: 175) GASASCAPSTG(SEQ ID NO: 176) GPEPTCPAPSG (SEQ ID NO: 177) GGSPAGSKTSP(SEQ ID NO: 178) GASASKAPSTG

In such cases where a gene encoding an XTEN with one or more cysteineand/or lysine motifs is to be constructed from existing XTEN modules,the gene can be designed and built by linking existing “building block”polynucleotides encoding both short- and long-length XTENs; e.g., AE48,AE144, AE288, AE576, AM48, AE864, AM875, AE912, AG864, or thenucleotides encoding the 36′mers of Examples 1-4, etc., which can befused in frame with the nucleotides encoding the cysteine- and/orlysine-containing motifs to build an engineered XTEN in which thereactive cysteine and/or lysines are placed in one or more selectedlocations in the sequence in the desired quantity. Non-limiting examplesof such engineered XTEN are provided in Table 8.

TABLE 8 Cysteine- and lysine- engineered XTEN SEQ XTEN ID Name*Amino Acid Sequence NO: AE144-GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG 179Island_Cys1-SEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGS AE576EPATSGSETPGTSTEPSEGSAPGGGSPAGSCTSPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG AE912-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEE 180Island_Cys2-GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG AE144TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGASASCAPSTGGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPG AE576-GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG 181Island_Cys1-TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT AE288SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGGGSPAGSCTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AE48-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGGPEPTCPAPS 182Island_Cys3-GGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA AE864PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTStEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AE288-GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG 183Island_Cys1-TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP AE288-AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSEIsland_Cys1-SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE AE288PSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGGSPAGSCTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGGSPAGSCTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AE48-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGGASASCAPST 184Island_Cys2-GGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA AE576-PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEIsland_Cys2-GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG AE144TSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGGASASCAPSTGGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSIEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSE GSAPGAM48- MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGGSPAGSCTS 185Island_Cys1-PGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSAS AM875PGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG AM48-MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGASASCAPST 186Island_Cys2-GGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSAS AM1296PGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG AM48-MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGPEPTCPAPS 187Island_Cys3-GGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSAS AM875PGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG AM48-MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGPEPTCPAPS 188Island_Cys3-GGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSAS AM875-PGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGIsland_Cys3-TSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT AM48STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGGPEPTCPAPSGGMAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPG AE144-GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG 189Island_Lys1-SEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGS AE576EPATSGSETPGTSTEPSEGSAPGGGSPAGSKTSPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG AE912-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEE 190Island_Lys2-GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG AE144TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGASASKAPSTGGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPG AE576-GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG 191Island_Lys1-TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT AE288SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGGGSPAGSKTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AE48-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGGASASKAPST 192Island_Lys2-GGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA AE864PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AE288-GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG 193Island_Lys1-TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP AE288-AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSEIsland_Lys1-SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE AE288PSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGGSPAGSKTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGGSPAGSKTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AE48-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGGGSPAGSKTS 194Island_Lys1-PGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA AE576-PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEIsland_Lys1-GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG AE144TSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGGGSPAGSKTSPGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGS APG AM48-MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGGSPAGSKTS 195Island_Lys1-PGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSAS AM875PGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG AM48-MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGASASKAPST 196Island_Lys2-GGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSAS AM1296PGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG AM48-MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGASASKAPST 197Island_Lys2-GGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSAS AM875PGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG AM48-MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGGASASKAPST 198Island_Lys2-GGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSAS AM875-PGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGIsland_Lys2-TSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT AM48STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGGASASKAPSTGGMAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPG AE288-GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG 199Island_Cys1-TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP AE288-AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSEIsland_Lys1-SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE AE288PSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGGSPAGSCTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGGGSPAGSKTSPGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AE48-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGGGSPAGSCTS 200Island_Cys1-PGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSA AE144-PGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPIsland_Cys1-GSEPATSGSETPGTSTEPSEGSAPGGGSPAGSCTSPGSEPATSGSETPGTSESATPESGPG AE144-SEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSIsland_Cys1-EPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGGG AE144-SPAGSCTSPGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSIsland_Cys1-TEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSE AE144SATPESGPGSEPATSGSETPGTSTEPSEGSAPGGGSPAGSCTSPGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS EGSAPGAE48- MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGGGSPAGSKTS 201Island_Lys1-PGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSA AE144-PGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPIsland_Lys1-GSEPATSGSETPGTSTEPSEGSAPGGGSPAGSKTSPGSEPATSGSETPGTSESATPESGPG AE144-SEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSIsland_Lys1-EPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGGG AE144-SPAGSKTSPGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSIsland_Lys1-TEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSE AE144SATPESGPGSEPATSGSETPGTSTEPSEGSAPGGGSPAGSKTSPGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS EGSAPG

In another embodiment, where an existing full-length XTEN gene is to bemodified with nucleotides encoding one or more reactive cysteine orlysine residues, an oligonucleotide can be created that encodes acysteine or lysine and that exhibits partial homology to and canhybridize with one or more short sequences of the XTEN, resulting in arecombination event and substitution of a cysteine or the lysine codonfor an existing codon of the XTEN gene (see, e.g., Example 61 for adescription of the general methods). In one embodiment, therecombination results in a replacement with the amino acid sequenceGGSPAGSCTSP. However, the oligonucleotides can be designed to place thecysteine (or lysine) in a different location in the motif or to includea second cysteine (or lysine) in the motif. The cysteine- orlysine-encoding oligonucleotides can be designed to hybridize with agiven sequence segment at different points along the known XTENsequence. Thus, the invention contemplates that multiple XTEN geneconstructs can be created with cysteines or lysines inserted atdifferent locations within the XTEN sequence by the selection ofrestriction sites within the XTEN sequence and the design ofoligonucleotides appropriate for the given location and that encode acysteine or lysine, including use of designed oligonucleotides thatresult in multiple insertions in the same XTEN sequence. By the designand selection of one or more such oligonucleotides in consideration ofthe known sequence of the XTEN, and the appropriate use of the methodsof the invention, the potential number of substituted reactive cysteineor lysine residues inserted into the full-length XTEN can be estimatedand then confirmed by sequencing the XTEN gene.

The design, selection, and preparation methods of the invention enablethe creation of engineered XTEN that are reactive with electrophilicfunctionality. These methods further enable creation of XTEN-drugconjugate compositions with drug molecules at designated, designed, andselective sites, as illustrated schematically in FIGS. 40-41. Drugs maybe site-specifically and efficiently coupled to cysteine-engineered XTENand targeted cysteine-engineered XTEN of the invention with athiol-reactive reagent. For example, reactive cysteine residues on acysteine-engineered XTEN allow specifically Connjugating a drug moietyto each cysteine of the XTEN sequence by cross-linking with a thiolreactive group such as maleimide or haloacetyl. FIG. 45 illustrates aspecific example of the conjugation of paclitaxel to an anti-Her2binding; fusion protein by this approach. Generally, the nucleophilicreactivity of the thiol functionality of a cysteine residue to amaleimide group is about 1000 times higher compared to any other aminoacid functionality in a protein, such as amino group of lysine residuesor the N-terminal amino group. Thiol specific functionality iniodoacetyl and maleimide reagents may react with amine groups, huthigher pH (>9.0) and longer reaction times are required (Garman, 1997,Non-Radioactive Labelling: A Practical Approach, Academic Press.London). Typically, conjugation reactions with cysteine are suitablyperformed at a pH below about 7, using reaction temperatures in therange of from about 5 up to about 40° C., and preferably in the range offrom 10 up to 30° C. (see U.S. Pat. No. 6,048,720).

1. Drugs

The drugs to be incorporated into the BFP-D and XTEN-drug compositionsof the invention have one or more pharmacologic activities. The drugsmay include a cytotoxic or cytostatic agent (e.g., epaclitaxel,paclitaxel, docetaxel, doxetaxel, irinotecan, pemetrexed, chlorambucil,or gemcitabine), an anti-inflammatory agent, an opiod (e.g., morphine,oxycodone, hydromorphone) an analgesic, an anti-infective, or afluorophore such as a fluorescent dye like fluorescein or rhodamine, achelating agent for an imaging or radiotherapeutic metal, a peptidyl ornon-peptidyl label or detection tag. In particular, drugs that have ahigh incidence of side effects or toxicity, or those for whichlocalization at the site of disease or pathology is desired, arecontemplated for incorporation into the BFP-D or XTEN-drug conjugates ofthe invention.

Exemplary drugs for incorporation into the compositions of the inventionare set forth in the official United States Pharmacopeia, officialHomeopathic Pharmacopeia of the United States, or official NationalFormulary, in the Physician's Desk Reference (PDR) and in the OrangeBook maintained by the U.S. Food and Drug Administration (FDA).Preferred drugs are those having the needed reactive functional group orthose that can be readily derivatized to provide the reactive functionalgroup for conjugation and will retain at least a portion of thepharmacologic activity of the unconjugated drug when conjugated to XTEN.In one embodiment, the drug for conjugation to the subject XTEN orfusion proteins disclosed herein is an agent selected from Table 9, or apharmaceutically acceptable salt, acid or derivative thereof.

TABLE 9 Drugs for Conjugation to Engineered XTEN Drugs Erlotinib;Bortezomib; Fulvestrant; Sutent (SU11248), Letrozole; Imatinib mesylate;PTK787/ZK 222584; Oxaliplatin; 5-FU (5-fluorouracil), leucovorin,rapamycin; lapatinib; lonafarnib; sorafenib; gefitinib; thiotepa;cyclosphosphamide; busulfan; improsulfan; piposulfan; benzodopa;carboquone; meturedopa; uredopa; altretamine; triethylenemelamine;triethylenephosphoramide; triethylenethiophosphoramide;trimethylomelamine; bullatacin; bullatacinone; camptothecin; topotecan;bryostatin; callystatin; CC-1065; adozelesin; calicheamycin; auristatin;carzelesin; bizelesin; cryptophycins (particularly cryptophycin 1 andcryptophycin 8); dolastatin; duocarmycin; eleutherobin; pancratistatin;sarcodictyin; spongistatin; chlorambucil; chlornaphazine;cholophosphamide; estramustine; ifosfamide; mechlorethamine;mechlorethamine oxide hydrochloride; melphalan; novembichin;phenesterine; prednimustine; trofosfamide; uracil mustard; carmustine;chlorozotocin; fotemustine; lomustine; nimustine; ranimnustine;calicheamicin; dynemicin; dynemicin A; clodronate; esperamicin;neocarzinostatin chromophore; aclacinomysins, actinomycin; anthramycin;azaserine; bleomycin; cactinomycin; carabicin; carminomycin;carzinophilin; chromomycinis; dactinomycin; daunorubicin; detorubicin;6-diazo-5-oxo-L-norleucine; doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin); epirubicin; esorubicin;idarubicin; marcellomycin; mitomycin C; mycophenolic acid; nogalamycin;olivomycin; peplomycin; potfiromycin; puromycin; quelamycin;rodorubicin; streptonigrin; streptozocin; tubercidin; ubenimex;zinostatin; zorubicin; methotrexate; 5-fluorouracil (5-FU); fdenopterin;methotrexate; pteropterin; trimetrexate; fludarabine; 6-mercaptopurine;thiamiprine; thioguanine; ancitabine; azacitidine; 6-azauridine;carmofur; cytarabine; dideoxyuridine; doxifluridine; enocitabine;floxuridine; calusterone; dromostanolone propionate; epitiostanol;mepitiostane; testolactone; aminoglutethimide; mitotane; trilostane;frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate;an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidainine; maytansine; ansamitocins; mitoguazone; mitoxantrone;mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine;razoxane; rhizoxin; ribavirin; zidovudine; acyclovir; gangcyclovir;vidarabine; idoxuridine; trifluridine; foscarnet; amantadine;rimantadine; saquinavir; indinavir; ritonavir; alpha-interferons andother interferons; AZT; sizofuran; spirogermanium; tenuazonic acid;triaziquone; 2;2′,2″-trichlorotriethylamine; T-2 toxin; verracurin A;roridin A; anguidine); urethan; vindesine; dacarbazine; mannomustine;mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”);cyclophosphamide; thiotepa; taxoids; epaclitaxel; paclitaxel; docetaxel;doxetaxel; irinotecan; pemetrexed chloranbucil; gemcitabine;6-thioguanine; mercaptopurine; methotrexate; cisplati; carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid;capecitabine; lidocaine; bupivacaine; memantine; donepezil;rivastigmine; galantamine; morphine; oxycodone; hydromorphone;oxymorphone; metopon; apomorphine; normorphine; etorphine;buprenorphine; meperidine; lopermide; anileridine; ethoheptazine;piminidine; betaprodine; diphenoxylate; fentanil; sufentanil;alfentanil; remifentanil; levorphanol; dextromethorphan; phenazocine;pentazocine; cyclazocine; methadone; isomethadone; propoxyphene;naloxone; naltrexone; treprostinil; N-methylnaloxone;6-amino-14-hydroxy-17- allylnordesomorphine; naltrendol;,N-methylnaltrexone; nalbuphine; butorphanol; cyclazocine; pentazocine,;nalmephene; naltrindole; nor-binaltorphimine; oxilorphan;6-amino-6-desoxo-naloxone; pentazocine; levallorphanmethylnaltrexone;buprenorphine; cyclorphan; levalorphan; cyclosporine; cyclosporine A;mycophenylate mofetil; sirolimus; tacrolimus; prednisone; azathioprine;methotrexate; cyclophosphamide; prednisone; aminocaproic acid;chloroquine; hydroxychloroquine; dexamethasone; chlorambucil; danazol;bromocriptine

2. Conjugation: Cross-Linkers and Methods

The conjugation between the polypeptide (either an XTEN or a fusionpartner, such as a targeting moiety) and the drug compound, optionallythrough a cross-linker, may be done according to methods known in theart, e.g. as described by Bodanszky in Peptide Synthesis, John Wiley,New York, 1976 and in WO 96/12505; Harris and Zalipsky, eds.,Poly(ethylene glycol) Chemistry and Biological Applications, AZC,Washington; R. F. Taylor, (1991), “Protein immobilisation. Fundamentaland applications”, Marcel Dekker, N.Y.; S. S. Wong, (1992), “Chemistryof Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), “Immobilized Affinity Ligand Techniques”,Academic Press, N.Y.; as well as in U.S. Pat. Nos. 5,977,163, 6,262,107,6,441,025, 7,026,440, 7,329,721, 7,528,202, 7,579,444, 7,659,361 and7,851,437; U.S. Patent App. Publication Nos. 2002001628, 20020077290,20040157782, and 20050238649; and PCT Publication Nos. WO 99/49901, WO97/33552, WO 01/26693, and WO 01/70275, or by methods disclosed herein.The exemplary methods of the foregoing patents or references, or thosedescribed herein, may be applied generally to the various binding fusionproteins disclosed herein, resulting in the drug-binding fusion proteincompositions, or to XTEN solely, resulting in the XTEN-D and/or BFP-Dcompositions of the invention.

Typically, attachment of a drug to a protein or other surface isaccomplished using an activated drug derivative, that is to say, a drughaving at least one activated terminus suitable for reaction with anucleophilic center (e.g., lysine, cysteine and similar residues ofproteins). Drug molecules having activated end groups suitable forreaction with the amino groups of proteins include those with functionalgroups such as aldehydes (Harris, J. M., Herati, R. S., Polym Prepr.(Am. Chem. Soc., Div. Polym. Chem), 32(1), 154-155 (1991), mixedanhydrides, N-hydroxysuccinimide esters, carbonylimadazolides, andchlorocyanurates (Herman, S., et al., Macromol. Chem. Phys. 195, 203-209(1994)). Although many proteins have been shown to retain activityduring modification, in some instances, drug attachment through proteinamino groups can be undesirable, such as when derivatization of specificlysine residues inactivates the pharmacophore of the protein (Suzuki,T., et al., Biochimica et Biophysica Acta 788, 248-255 (1984)).Moreover, since many non-XTEN proteins possess severalavailable/accessible amino groups, the resulting drug conjugates formedare typically mixtures of mono-, di-, tri-conjugated species and so on,which can be difficult and also time-consuming to characterize andseparate. One method for avoiding these problems is to employ asite-selective reagent that targets functional groups other than aminesOne particularly attractive target is the thiol group, which in proteinsin present in the amino acid, cysteine. Cysteines are typically lessabundant in proteins than lysines, thus reducing the likelihood ofprotein deactivation upon conjugation to these thiol-containing aminoacids. Moreover, conjugation to cysteine sites can often be carried outin a well-defined manner, leading to the formation of single speciespolymer-conjugates.

The thiol-reactive reagent may be a multifunctional cross-linkerreagent, a drug-linker, a capture reagent, i.e. affinity moiety, labelreagent (e.g. a biotin-linker reagent), a detection label (e.g. afluorophore reagent), a solid phase immobilization reagent (e.g.SEPHAROSE™, polystyrene, or glass), or a drug-cross-linker intermediate.One example of a thiol-reactive reagent is N-ethyl maleimide (NEM). Inan exemplary embodiment, reaction of a thiol-XTEN with a biotin-linkerreagent provides a biotinylated thiol-XTEN by which the presence andreactivity of the engineered cysteine residue may be detected andmeasured. Reaction of a thiol-XTEN with a multifunctional cross-linkerreagent provides a thiol-XTEN with a functionalized cross-linker thatmay be further reacted with a drug moiety reagent or other label. In oneembodiment, reaction of a thiol-XTEN or a targeted thiol-XTEN with adrug-linker intermediate provides a thiol-XTEN drug conjugate or atargeted thiol-XTEN drug conjugate, respectively.

A variety of linkage chemistries can be used for conjugation, includingcommercially available homo- or hetero-bifunctional cross-linkercompounds, according to methods known and available in the art, such asthose described, for example, in Hermanson, Greg T., BioconjugateTechniques, Academic Press, Inc., 1995, and Wong, Shan S., Chemistry.Suitable cross-linking agents for use in preparing the compositions ofthe disclosure are commercially available from companies likeSigma-Aldrich, or Thermo Fisher Scientific Inc. (Pierce Protein ResearchProducts). Of particular utility are cross-linker components that areavailable in activated form and can be directly used for conjugation.Examples of useful cross-linking agents are imidoesters, activehalogens, maleimide, pyridyl disulfide, and NHS-esters. Homobifunctionalcross-linking agents have two identical reactive groups and are oftenused in a one step chemical cross-linking procedure. Examples are BS3 (anon-cleavable water-soluble DSS analog), BSOCOES (base-reversible), DMA(Dimethyl adipimidate-2HCl), DMP (Dimethyl pimelimidate-2HCl), DMS(Dimethyl suberimidate-2HCl), DSG (5-carbon analog of DSS), DSP(Lomant's reagent), DSS (non-cleavable), DST (cleavable by oxidizingagents), DTBP (Dimethyl 3,3′-dithiobispropionimidate-2HCl), DTSSP, EGS,Sulfo-EGS, THPP, TSAT, PMPI (N-[p-maleimidophenyl]isocyanate), DFDNB(1,5-Difluoro-2,4-dinitrobenzene) is especially useful for cross-linkingbetween small spacial distances (Komblatt, J. A. and Lake, D. F. (1980).Cross-linking of cytochrome oxidase subunits withdifluorodinitrobenzene. Can J. Biochem. 58, 219-224).

Sulfhydryl-reactive homobifunctional cross-linking agents arehomobifunctional protein cross-linkers that react with sulfhydryls andare often based on maleimides and maleamic acid, which react with —SHgroups, forming stable thioether linkages. The reaction can be conductedat a pH of from about 6 to about 10, and at, for example, a pH of about6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10, with optimal pH at about6.5 to about 8. In embodiments based on maleimide cross-linkers, one canfurther increase the stability of conjugates the maleimide ring can beintentionally forced open by hydrolysis during the reaction using a pHof 7 to about 9 and reaction temperature of about 10° C. to 45° C., orabout 18° C. to about 30° C., to provide conjugates where the maleimidecomponent is converted to its more stable succinamic acid opened-ringform.

For drugs with conjugated maleimide cross-linkers, the method includesreacting the protein with an active agent that possesses a nucleophileunder conditions effective to couple the drug-linker to the protein. Anexample of such a cross-linker and reaction include BM[PEO]3; an 8-atompolyether spacer that reduces potential for conjugate precipitation insulfydryl-to-sulfhydryl cross-linking applications. BM[PEO]4 is similarbut with an 11-atom spacer. BMB is a non-cleavable cross-linker with afour-carbon spacer. BMDB makes a linkage that can be cleaved withperiodate. BMH is a widely used homobifunctional sulfhydryl-reactivecross-linker. BMOE has an especially short cross-linker. DPDPB and DTMEare cleavable cross-linkers. HVBS does not have the hydrolysis potentialof maleimides. TMEA is another option. Hetero-bifunctional cross-linkingagents have two different reactive groups. Examples are NHS-esters andamines/hydrazines via EDC activation, AEDP, ASBA (photoreactive,iodinatable), EDC (water-soluble carbodiimide) Amine-Sulfhydryl reactivebifunctional cross-linkers are AMAS, APDP, BMPS, EMCA, EMCS, GMBS, KMUA,LC-SMCC, LC-SPDP, MBS, SBAP, SIA (extra short), SIAB, SMCC, SMPB, SMPH,SMPT, SPDP, Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-LC-SMPT,Sulfo-LC-SPDP, Sulfo-MBS, Sulfo-SIAB, Sulfo-SMCC, Sulfo-SMPB.Sulfhydryl-carbonyl reactive bifunctional cross linkers, such as KMUH(N-[k-Maleimidoundecanoic acid]hydrazide), BMPH (N-[ß-Maleimidopropionicacid]hydrazide), EMCH ([N-e-Maleimidocaproic acid]hydrazide), MPBH(4-(4-N-Maleimidophenyl)butyric acid hydrazide hydrochloride), and PDPH(3-(2-Pyridyldithio)propionyl hydrazide) Amino-group reactiveheterobifunctional cross-linking agents are ANB-NOS, MSA, NHS-ASA, SADP,SAED, SAND, SANPAH, SASD, SFAD, Sulfo-HSAB, Sulfo-NHS-LC-ASA,Sulfo-SADP, Sulfo-SANPAH, TFCS. Arginine-reactive cross-linking agentsare, for example APG, which reacts specifically with arginines at pH7-8.

For drugs with conjugated maleimide cross-linkers, the method includesreacting the protein with an active agent that possesses a nucleophileunder conditions effective to couple the drug-linker to the MichaelAddition Receptor protein via a Michael-type addition reaction to form apolymer-succinimide-linked protein-drug conjugate. A “Michael AdditionReceptor”, as one skilled in the art will understand, is a moietycapable of reacting with a nucleophilic reagent so as to undergo anucleophilic addition reaction characteristic of a Michael Additionreaction. After the nucleophilic addition occurs, the Michael AdditionReceptor moiety is referred to as a “Michael Addition Adduct.”Typically, a Michael Addition is the nucleophilic addition of acarbanion or another nucleophile to an alpha, beta unsaturated carbonylcompound, such as a thioacid that is created by treating the cysteineresidues of the engineered XTEN to obtain a thiol, then a thioacid.Alternatively, the epsilon amino groups of the lysine-engineered XTENcan be thioloated using thiolating reagents, for example, SPDP oriminothiolane, to create the Michael Addition Receptor. Such methods areknown in the art (see, e.g., U.S. Pat. No. 5,708,146).

Functional groups on the drug to be conjugated can include carboxylicacid functional groups and chloroformate functional groups, which areuseful reactive sites because they can react with epsilon amino groupsof a lysine-engineered XTEN or a cross-linker to form an amide linkage.Also useful as a reactive site is a carbonate functional group, such asbut not limited to p-nitrophenyl carbonate, which can react with anamino group to form a carbamate linkage. Where the drug is to beconjugated through a hydroxyl group to the XTEN, the hydroxyl end groupsof the drug molecule must be modified and/or provided in activated form,i.e. with reactive functional groups (examples of which include primaryamino groups, aminoxy, aldehyde, hydrazide (HZ), thiol, thiolate,succinate (SUC), succinimidyl succinate (SS), succinimidyl succinate“active ester”, succinimidyl succinamide (SSA), succinimidyl propionate(SPA), succinimidyl butanoate (SBA), succinimidyl carboxymethylate(SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS),aldehyde, nitrophenylcarbonate (NPC), and tresylate (TRES)). Othersuitable reactive functional groups of drug molecules include acetal,aldehydes having a carbon length of 1 to 25 carbons (e.g., acetaldehyde,propionaldehyde, and butyraldehyde), aldehyde hydrate, alkenyl,acrylate, methacrylate, acrylamide, active sulfone, amine, hydrazide,thiol, alkanoic acids (e.g., carboxylic acid, carboxymethyl, propanoicacid, and butanoic acid), acid halide, isocyanate, isothiocyanate,maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide,epoxide, glyoxal, dione, mesylate, tosylate, and tresylate.

The drug can also be conjugated using a heterocycle radical of a ringsystem. Heterocyclyl groups include a ring system in which one or morering atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 1 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6]system. Heterocycles are described in Paquette, Leo A.; “Principles ofModern Heterocyclic Chemistry” (W. A. Benjamin, New York, 1968); “TheChemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley& Sons, New York, 1950 to present), in particular Volumes 13, 14, 16,19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. Examples ofheterocycles that may be found in drugs suitable for conjugation includeby way of example and not limitation pyridyl, dihydroypyridyl,tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, sulfuroxidized tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl,pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl,indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl,piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl,tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl,bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl,6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl,pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl,2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4Ah-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

In some cases, the drug molecules are attached to the lysine- orcysteine engineered XTEN by cross-linkers having two reactive sites forbinding to the drug and the XTEN. Preferred cross-inker groups are thosethat are relatively stable to hydrolysis in the circulation, arebiodegradable and are nontoxic when cleaved from the conjugate. Inaddition, the use of cross-linkers can provide the potential forconjugates with an even greater flexibility between the drug and theXTEN, or provide sufficient space between the drug and the XTEN suchthat the XTEN does not interfere with the binding between thepharmacophore and its binding site. In one embodiment, a cross-linkerhas a reactive site that has an electrophilic group that is reactive toa nucleophilic group present on an XTEN. Preferred nucleophiles includethiol, thiolate, and amino. The heteroatom of the nucleophilic group ofa lysine- or cysteine-engineered XTEN is reactive to an electrophilicgroup on a cross-linker and forms a covalent bond to the cross-linkerunit. Useful electrophilic groups for cross-linkers include, but are notlimited to, maleimide and haloacetamide groups, and provide a convenientsite for attachment to the XTEN

In another embodiment, a cross-linker has a reactive site that has anucleophilic group that is reactive to an electrophilic group present ona drug. Useful electrophilic groups on a drug include, but are notlimited to, hydroxyl, thiol, aldehyde and ketone carbonyl groups. Theheteroatom of a nucleophilic group of a cross-linker can react with anelectrophilic group on a drug and form a covalent bond. Usefulnucleophilic groups on a cross-linker include, but are not limited to,hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazinecarboxylate, and arylhydrazide. The electrophilic group on a drugprovides a convenient site for attachment to a cross-inker.

For conjugation of drugs to the lysine epsilon amino group oflysine-engineered XTEN, use of reactive drug-N-hydroxylsuccinimide, oresters such as drug-succinimidyl propionate, or drug-succinimidylbutanoate or other drug-succinimide conjugates can be employed.Alternatively, lysine residues may be used to introduce free sulfhydrylgroups through reaction with iminothiolane. Alternatively, targetingsubstance lysines may be linked to a heterobifunctional reagent having afree hydrazide or aldehyde group available for conjugation with anactive agent. Reactive esters can couple at physiological pH, but lessreactive derivatives typically require higher pH. Low temperatures mayalso be employed if a labile protein is being used. Under lowtemperature conditions, a longer reaction time may be used for theconjugation reaction.

Amino group conjugation with lysine residues is facilitated by thedifference between the pKa values of the α-amino group of the N-terminalamino acid (approximately 7.6 to 8.0) and the ε-amino group of lysine(approximately 10). Conjugation of the terminal amino group oftenemploys reactive drug-aldehydes (such as drug-propionaldehyde ordrug-butylaldehyde), which are more selective for amines and thus areless likely to react with, for example, the imidazole group ofhistidine. In addition, lysinyl amino residues are reacted with succinicor other carboxylic acid anhydrides, or with N,N′-Disuccinimidylcarbonate (DSC), N,N′-carbonyl diimidazole (CDI), or p-nitrophenylchloroformate to yield the activated succinimidyl carbonate, imidazolecarbamate or p-nitrophenyl carbonate, respectively. Derivatization withthese agents has the effect of reversing the charge of the lysinylresidues. Conjugation of a drug-aldehyde to the terminal amino grouptypically takes place in a suitable buffer performed at a pH whichallows one to take advantage of the pKa differences between the ε-aminogroups of the lysine residues and that of the α-amino group of theN-terminal residue of the protein; usually the pH for coupling lies inthe range of from about pH 7 up to about 8. Useful methods forconjugation of the lysine epsilon amino group have been described inU.S. Pat. Nos. 4,904,584 and 6,048,720.

The person with ordinary skill in the art will be aware that theactivation method and/or conjugation chemistry to be used depends on thereactive groups of the XTEN polypeptide as well as the functional groupsof the drug moiety (e.g., being amino, hydroxyl, carboxyl, aldehyde,sulfhydryl, etc), the functional group of the drug-cross-linkerreactant, or the functional group of the XTEN-cross-linker reactant. Thedrug conjugation may be directed towards conjugation to all availableattachment groups on the engineered XTEN polypeptide such as thespecific engineered attachment groups on the incorporated cysteineresidues or lysine residues. In order to control the reactants such thatthe conjugation is directed to the appropriate reactive site, theinvention contemplates the use of protective groups. A “protectinggroup” is a moiety that prevents or blocks reaction of a particularchemically reactive functional group in a molecule under certainreaction conditions. The protecting group will vary depending upon thetype of chemically reactive group being protected as well as thereaction conditions to be employed, as well as the presence ofadditional reactive groups in the molecule. Non-limiting examples offunctional groups which may be protected include carboxylic acid groups,hydroxyl groups, amino groups, hydroxyl groups, thiol groups, andcarbonyl groups. Representative protecting groups for carboxylic acidsand hydroxyls include esters (such as a p-methoxybenzyl ester), amidesand hydrazides; for amino groups, carbamates (such astert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters;for thiol groups, thioethers and thioesters; for carbonyl groups,acetals and ketals; and the like. Such protecting groups are well-knownto those skilled in the art and are described, for example, in T. W.Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, ThirdEdition, Wiley, New York, 1999, and references cited therein.

The conjugation may be achieved in one step or in a stepwise manner(e.g., as described in WO 99/55377), such as through addition of areaction intermediate cross-linker, using the cross-linkers disclosedherein or those known in the art to be useful for conjugation tocysteine or lysine residues of polypeptides to be linked to reactivefunctional groups on drug molecules.

In some cases, the method for conjugating a cross-linker to acysteine-engineered XTEN may provide that the XTEN is pre-treated with areducing agent, such as dithiothreitol (DTT) to reduce any cysteinedisulfide residues to form highly nucleophilic cysteine thiol groups(—CH₂SH). The reducing agent is subsequently removed by any conventionalmethod, such as by desalting. The partially reduced XTEN thus reactswith drug-linker compounds, or cross-linker reagents, with electrophilicfunctional groups such as maleimide or α-halo carbonyl, according to,for example, the conjugation method of Klussman, et al. (2004),Bioconjugate Chemistry 15(4):765-773. Conjugation of a cross-linker or adrug to a cysteine residue typically takes place in a suitable buffer atpH 6-9 at temperatures varying from 4° C. to 25° C. for periods up toabout 16 hours. Alternatively, the cysteine residues can be derivatizedwith an organic derivatizing agent. Suitable derivatizing agents andmethods are well known in the art. For example, cysteinyl residues mostcommonly are reacted with α-haloacetates (and corresponding amines),such as chloroacetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteinyl residues also are derivatizedby reaction with bromotrifluoroacetone, α-bromo-β-(4-imidozoyl)propionicacid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

In one embodiment, XTEN can be dissolved in 500 mM sodium borate and 500mM sodium chloride at pH 8.0 and then is treated with an excess of 100mM dithiothreitol (DTT). After incubation at 37° C. for about 30minutes, the buffer is exchanged by elution over Sephadex G25 resin andeluted with PBS with 1 mM DTPA. The thiol/XTEN value is checked bydetermining the reduced XTEN concentration from the absorbance at 280 nmof the solution and the thiol concentration by reaction with DTNB(Aldrich, Milwaukee, Wis.) and determination of the absorbance at 412nm. The reduced XTEN dissolved in PBS is chilled on ice. The drugcross-linker, e.g., MC-val-cit-PAB-MMAE in DMSO, dissolved inacetonitrile and water at known concentration, is added to the chilledreduced XTEN in PBS. After about one hour, an excess of maleimide isadded to quench the reaction and cap any unreacted antibody thiolgroups. The reaction mixture is concentrated by centrifugalultrafiltration and the XTEN-MC-vc-PAB-MMAE, is purified and desalted byelution through G25 resin in PBS, filtered through 0.2 μm filters understerile conditions, and held under suitable storage conditions.

Such an approach may used to conjugate other thiol-reactive agents tothe cysteine of the XTEN, in which the reactive group is, for example, amaleimide, an iodoacetamide, a pyridyl disulfide, or otherthiol-reactive conjugation partner linked to a drug partner (Haugland,2003, Molecular Probes Handbook of Fluorescent Probes and ResearchChemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem.3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach,Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson,G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp.40-55, 643-671). Maleimides in particular are useful in cross-linkingdue to their susceptibility to additions across the double bond eitherby Michael additions or via Diels-Alder reactions. Bismaleimides are aclass of maleimide compounds with two maleimide groups connected througha molecular unit and can be used as cross-linking reagents.

In some instances, the conjugation is performed under conditions aimingat reacting as many of the available XTEN attachment groups as possiblewith drug or drug-linker molecules. This is achieved by means of asuitable molar excess of the drug in relation to the polypeptide.Typical molar ratios of activated drug or drug-linker molecules topolypeptide are up to about 1000-1, such as up to about 200-1 or up toabout 100-1. In some cases, the ratio may be somewhat lower, however,such as up to about 50-1, 10-1 or 5-1. Also equimolar ratios may beused.

In some case, the drug-containing conjugate compositions of thedisclosure retain at least a portion of the pharmacologic activitycompared to the corresponding unconjugated drug. In one embodiment, thedrug conjugate retains at least about 1%, or at least about 5%, or atleast about 10%, or at least about 20%, or at least about 30%, or atleast about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90%, or atleast about 95% of the pharmacologic activity of the unconjugated drug.

In other cases, the drug may be derivatized through a reactivefunctional group that is important for the biological activity of thedrug thereby inhibiting or reducing the pharmacological activity of thedrug to thereby convert the drug into a pharmacologically inactive orrelatively inactive peptidyl derivative conjugate. In one embodiment,the prodrug cross-linker contains a peptide residue specificallytailored so as to render a drug conjugate of the present invention aselective substrate susceptible to enzymatic cleavage by one or moreproteases, e.g., preferably lysosomal proteases, such as cathepsin B, Cor D. The enzymatic cleavage reaction will remove the prodrugcross-linker from the drug moiety and affect the release of the drug inits pharmacologically active form. Representative hydrolyticallydegradable linkages in a cross-linker-drug conjugate include carboxylateester, carbonate ester, phosphate ester, anhydride, acetal, ketal,acyloxyalkyl ether, imine, orthoester, and oligonucleotides. Esters suchas carboxylate and carbonate esters are particularly preferred linkages.The particular linkage and linkage chemistry employed will depend uponthe particular active agent, the presence of target and additionalfunctional groups within the active agent, and the like; considerationsthat are within the knowledge of one skilled in the art. By such anapproach, the inventive XTEN-drug and binding fusion protein-drugconjugate compositions administered to a subject may exhibit reducedtoxicity or frequency of side effects compared to the corresponding freedrug administered to a subject. Thus, the reduced toxicity of theXTEN-drug and binding fusion protein-drug conjugate compositionsdisclosed herein may permit the administration of higher amounts ofdrug, on a molar basis, compared to unconjugated drug.

The invention contemplates that the engineered XTEN, which incorporateeither cysteine or lysine residues, may be conjugated with any drugmoiety with a reactive functional group that can be covalently attachedto the XTEN through a reactive cysteine thiol or epsilon amino group,respectively, either directly or by using a cross-linker, as describedabove. In another embodiment, the invention provides BFP-D compositionsin which the drug can be conjugated to the targeting moiety of thebinding protein component by conjugation to existing or incorporatedcysteine or lysine residues, as well as N-terminal amino groups orC-terminal carboxyl groups that may be present.

(h) Release of Drug

The invention provides BFP-D and XTEN-drug compositions in which thedrug can be released from the composition by either specific ornon-specific mechanisms. In some cases, the drugs can be released byproteolytic degradation of those molecules taken up by cells. In oneembodiment, the XTEN portion of the BFP-D or XTEN-drug is rapidlydegraded by intracellular proteases, releasing the drug from the XTENcarrier. In other cases, the drug can be released by degradation of across-linker selected for inclusion into the BFP-D and XTEN-drugcompositions based on its susceptibility to degradation. For example, itis known in the art that use of mild acid-cleavable linkers can promotedrug release based on the observation that the pH inside tumors wasoften lower than normal physiological pH. In a non-limiting example,release of the conjugated drug component could be enhanced byincorporating a hydrazone as a cleavable unit and attaching a drug likedoxorubicin to the protein component (either XTEN or the targetingmoiety) via a thioether group, as described by Willner et al., U.S. Pat.No. 5,708,146; and Trail et al. Cure of xenografted human carcinomas byBR96-doxorubicin immunoconjugates. Science 261:212-215 (1993). In othercases, certain ester linkages can be incorporated into the linkerbetween a protein and the drug that are labile; some by enzymes.(Gillimard and Saragovi, Cancer Res. 61:694-699 (2001)). Other examplesof enzymatically susceptible linkages include urethane orcarbonate-containing linkages. In addition, hydrolytically unstablelinkages include carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides andoligonucleotides. Based upon these characteristics, it is believed thatthe drug ligand will be released rapidly after cellular internalizationof the BFP-D or XTEN-drug conjugates.

(i) Configurations of BFP-D and XTEN-Drug Compositions

The invention provides binding fusion protein-drug conjugates andXTEN-drug conjugates in various configurations.

In one embodiment, the invention provides a binding fusion protein-drugconjugate composition of formula V:[(D-CL)_(z1)-XTEN]_(x)-TM-[XTEN-(CL-D)_(z2)]_(y)  Vwherein independently for each occurrence: x is either 0 or 1; y iseither 0 or 1; XTEN is a cysteine- or lysine-engineered extendedrecombinant polypeptide as described above; TM is a targeting moietywith binding affinity to a target ligand selected from Table 1 or Table2 (which may comprise more than one binding domain joined by linkers);CL is a cross-linker as defined herein; D is a drug moiety selected fromTable 9 or a pharmaceutically acceptable salt, acid or derivativethereof; and z1 and z2 each independently is an integer from 1 to 100.The number of drug moieties that may be conjugated via a reactivecross-linker to an engineered XTEN molecule is limited by the number ofreactive residues that are incorporated into the XTEN. Exemplary bindingfusion protein-drug conjugate compositions of Formula V can compriseXTEN that have from 1 to about 100 cysteine or lysine engineered aminoacids, or from 1 to about 50 cysteine or lysine engineered amino acids,or from 1 to about 40 cysteine or lysine engineered amino acids, or from1 to about 20 cysteine or lysine engineered amino acids, or from 1 toabout 10 cysteine or lysine engineered amino acids, or from 1 to about 5cysteine or lysine engineered amino acids that are available forconjugation to drug molecules. In some cases, the binding fusionprotein-drug conjugate retains at least about 1%, or at least about 5%,or at least about 10%, or at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90%, or atleast about 95% of the pharmacologic activity of the unconjugated drug.

In another embodiment, the invention provides a binding fusionprotein-drug conjugate composition of formula VI:[(D-CL)_(z1)-XTEN]_(x)-TM1-L-TM2-[XTEN-(CL-D)_(z2)]_(y)  VIwherein independently for each occurrence: x is either 0 or 1, and y iseither 0 or 1; XTEN is a either a cysteine- or lysine-engineeredextended recombinant polypeptide as described; TM1 is a targeting moietywith binding affinity to a target ligand selected from Table 1 or Table2 (which may comprise more than one binding domain joined by linkers);TM2 is a targeting moiety with binding affinity to a target ligandselected from Table 1 or Table 2 (which may comprise more than onebinding domain joined by linkers) that may be identical or may bedifferent to TM1; and L is a linker sequence having between 1 to about300 amino acid residues wherein the linker sequence is covalently boundto the C terminus of TM1 and the N terminus of TM2; D is a drug moietyselected from Table 9 or a pharmaceutically acceptable salt, acid orderivative thereof; CL is a cross-linker as defined herein; and z1 andz2 each independently is an integer from 0 to 100. Exemplary bindingfusion protein-drug conjugate compositions of Formula VI can compriseXTEN that have from 1 to about 100 cysteine or lysine engineered aminoacids, or from 1 to about 50 cysteine or lysine engineered amino acids,or from 1 to about 40 cysteine or lysine engineered amino acids, or from1 to about 20 cysteine or lysine engineered amino acids, or from 1 toabout 10 cysteine or lysine engineered amino acids, or from 1 to about 5cysteine or lysine engineered amino acids that are available forconjugation to drug molecules. In addition, the invention contemplatesadditional compositions comprising multiple targeting moieties and XTENin various permutations of configurations. In some cases, the bindingfusion protein-drug conjugate retains at least about 1%, or at leastabout 5%, or at least about 10%, or at least about 20%, or at leastabout 30%, or at least about 40%, or at least about 50%, or at leastabout 60%, or at least about 70%, or at least about 80%, or at leastabout 90%, or at least about 95% of the pharmacologic activity of theunconjugated drug.

In another embodiment, the invention provides a pharmacologically activeXTEN-drug conjugate composition of formula VII:(CL-D)_(z)-XTEN  VIIwherein independently for each occurrence: XTEN is a either a cysteine-or lysine-engineered extended recombinant polypeptide as describedabove; D is a drug moiety selected from Table 9 or a pharmaceuticallyacceptable salt, acid or derivative thereof; CL is a cross-linker asdefined herein; and z is an integer from 1 to 100. Exemplary XTEN-drugconjugate compositions of formula VII can comprise XTEN that have from 1to about 100 cysteine or lysine engineered amino acids, or from 1 toabout 50 cysteine or lysine engineered amino acids, or from 1 to about40 cysteine or lysine engineered amino acids, or from 1 to about 20cysteine or lysine engineered amino acids, or from 1 to about 10cysteine or lysine engineered amino acids, or from 1 to about 5 cysteineor lysine engineered amino acids that are available for conjugation todrug molecules. In some cases, the XTEN-drug conjugate retains at leastabout 1%, or at least about 5%, or at least about 10%, or at least about20%, or at least about 30%, or at least about 40%, or at least about50%, or at least about 60%, or at least about 70%, or at least about80%, or at least about 90%, or at least about 95% of the pharmacologicactivity of the unconjugated drug.

In another embodiment, the invention provides a pharmacologically activeXTEN-drug conjugate composition of formula VIII:XTEN-(CL-D)_(z)  VIIIwherein independently for each occurrence: XTEN is a either a cysteine-or lysine-engineered extended recombinant polypeptide as describedabove; D is a drug moiety selected from Table 9 or a pharmaceuticallyacceptable salt, acid or derivative thereof; CL is a cross-linker asdefined herein; and z is an integer from 1 to 100. Exemplary XTEN-drugconjugate compositions of formula VII can comprise XTEN that have from 1to about 100 cysteine or lysine engineered amino acids, or from 1 toabout 50 cysteine or lysine engineered amino acids, or from 1 to about40 cysteine or lysine engineered amino acids, or from 1 to about 20cysteine or lysine engineered amino acids, or from 1 to about 10cysteine or lysine engineered amino acids, or from 1 to about 5 cysteineor lysine engineered amino acids that are available for conjugation todrug molecules. In some cases, the XTEN-drug conjugate retains at leastabout 1%, or at least about 5%, or at least about 10%, or at least about20%, or at least about 30%, or at least about 40%, or at least about50%, or at least about 60%, or at least about 70%, or at least about80%, or at least about 90%, or at least about 95% of the pharmacologicactivity of the unconjugated drug.

The invention contemplates that the BFP-D encompass compositions inwhich the targeting moiety component(s) of the subject compositions canbe directed to any of the specific targets described herein, including atarget selected from Table 1 or Table 2, the drug conjugated to thecomposition can be any of the drugs of Table 9 or a pharmaceuticallyacceptable salt, acid or derivative thereof, and the XTEN component(s)can be cysteine- or lysine-engineered XTEN derived from or exhibitingsubstantial sequence identity to any of the XTEN of Table 4 or afragment or variant thereof, and has cross-linker components that linkthe drug molecule(s) to the protein component. In an embodiment of theforegoing, the BFP-D composition can have one or more engineered-XTENsin which the XTEN has about 80% sequence identity to a XTEN selectedfrom Table 4, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequenceidentity to an XTEN selected from Table 4 and comprises one or morecysteine residues. In another embodiment of the foregoing, the BFP-Dcomposition can have one or more engineered-XTENs in which the XTEN hasabout 80% sequence identity to a XTEN selected from Table 4, oralternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity to anXTEN selected from Table 4 and comprises one or more lysine residues. Inany of the embodiments hereinabove described in this paragraph, theengineered XTEN can exhibit about 80% sequence identity to a XTENselected from Table 8, or alternatively 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, orexhibit 100% sequence identity to an XTEN selected from Table 8.

In an exemplary embodiment of a BFP-D, the binding fusion protein-drugcomposition comprises one or more anti-HER2 targeting moieties, anengineered XTEN, and one or more molecules of paclitaxel linked to theXTEN by a cross-linker. In another embodiment, the binding fusionprotein-drug composition comprises one or more anti-HER2 targetingmoieties, an engineered XTEN, and one or more molecules of docetaxellinked to the XTEN by a cross-linker. In another embodiment, the bindingfusion protein-drug composition comprises one or more anti-HER2targeting moieties, an engineered XTEN, and one or more molecules ofirinotecan linked to the XTEN by a cross-linker. In any of the foregoingembodiments of the paragraph, the invention encompasses compositionsthat can be configured according to formula V or formula VI.

In an exemplary embodiment of an XTEN-D, the XTEN-drug compositioncomprises an engineered XTEN and one or more molecules of paclitaxellinked to the XTEN by a cross-linker. In another embodiment, theXTEN-drug composition comprises an engineered XTEN and one or moremolecules of docetaxel linked to the XTEN by a cross-linker. In anotherembodiment, the XTEN-drug composition comprises an engineered XTEN andone or more molecules of irinotecan linked to the XTEN by across-linker. In any of the foregoing embodiments of the paragraph, theinvention encompasses compositions that can be configured according toformula VI or formula VII.

Generally, binding fusion protein-drug conjugate compositions of theinvention retain the antigen binding capability of their targetingmoiety. In some cases, wherein the drugs are restricted to the XTENcarrier portion of the fusion protein, steric hindrance between the drugmoiety, targeting moiety, and the target antigen or ligand is reduced.Thus, in preferred embodiments, engineered binding fusion protein-drugconjugates are capable of binding, preferably specifically, to targetantigens and delivering the drug to the target location. Such antigensinclude, for example, tumor-associated antigens (TAA), cell surfacereceptor proteins and other cell surface molecules, transmembraneproteins, signaling proteins, cell survival regulatory factors, cellproliferation regulatory factors, molecules associated with tissuedevelopment or differentiation, lymphokines, cytokines, moleculesinvolved in cell cycle regulation, molecules involved in vasculogenesisor angiogenesis. An antigen to which an engineered binding fusionprotein-drug composition is capable of binding may be a member of asubset of one of the above-mentioned categories, wherein the othersubset(s) of said category comprise other molecules/antigens that have adistinct characteristic (with respect to the antigen of interest).

The invention also contemplates that the XTEN-drug encompasscompositions in which the drug conjugated to the XTEN can be any of thedrugs of Table 9 or a pharmaceutically acceptable salt, acid orderivative thereof, and the XTEN component can be cysteine- orlysine-engineered XTEN derived from or exhibiting substantial sequenceidentity to any of the XTEN of Table 4 or a fragment or variant thereof,and has cross-linker components that link the drug molecule(s) to theXTEN component. In an embodiment of the foregoing, the XTEN-drugcomposition can have an engineered-XTENs in which the XTEN has about 80%sequence identity to a XTEN selected from Table 4, or alternatively 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or about 99% sequence identity to an XTEN selected fromTable 4 and comprises one or more cysteine residues. In anotherembodiment of the foregoing, the XTEN-drug composition can have anengineered-XTEN in which the XTEN has about 80% sequence identity to aXTEN selected from Table 4, or alternatively 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, orabout 99% sequence identity to an XTEN selected from Table 4 andcomprises one or more lysine residues. In some cases, the XTEN-drugencompass compositions in which the drug conjugated to the XTEN can beany of the drugs of Table 9 or a pharmaceutically acceptable salt, acidor derivative thereof, and the XTEN component can be cysteine- orlysine-engineered XTEN derived from or exhibiting substantial sequenceidentity to any of the engineered XTEN of Table 8 or a fragment orvariant thereof, and has cross-linker components that link the drugmolecule(s) to the cysteine or lysine residues of the XTEN component. Inone embodiment of the foregoing, the engineered XTEN can exhibit about80% sequence identity to a XTEN selected from Table 8, or alternatively81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or exhibits 100% sequence identity to an XTENselected from Table 8.

(j) Methods of Use of BFP-Drug and XTEN-Drug Compositions

In another aspect, the invention provides a method of for achieving abeneficial effect in a disease, disorder or condition mediated by aBFP-D or XTEN-drug composition. In one embodiment, the inventionprovides the use of a BFP-D, in which the targeting moiety of thebinding fusion protein is derived from a parental antibody that binds toa target selected from the group consisting of the targets of Table 1 orTable 2 and the drug is selected from Table 9, in treatment of adisease, disorder or condition to a subject in need thereof byadministration of a therapeutically effective amount of the BFP-D,wherein said administration leads to the eradication or amelioration ofone or more of the physiological or clinical symptoms associated withthe underlying disorder such that an improvement is observed in thesubject, notwithstanding that the subject may still be afflicted withthe underlying disorder. In another embodiment, the invention provides amethod of treating a disease, disorder, or condition in a mammalcomprising administering to the mammal a therapeutically effectiveamount of a BFP-D comprising one or more targeting moieties directed toone or more targets selected from Table 1 or Table 2, linked to one ormore XTEN sequences molecules and, optionally, one or more linkers, toform the binding fusion protein component, wherein the linkage does notsubstantially alter the essential functional properties of bindingaffinity and sustained terminal half-life or reduced serum clearancerate as compared to that of the parental targeting moiety from which thebinding fusion protein component is derived and wherein the drugconjugated to the BFP-D is selected from Table 9, wherein theadministration of the BFP-D to a subject in need thereof achieves abeneficial therapeutic effect. The effective amount can produce abeneficial effect in helping to treat (e.g., cure or reduce theseverity) or prevent (e.g., reduce the likelihood of onset or severity)a disease, disorder or condition, such as, but not limited to a cancer,a cardiovascular disease or condition, an infectious disease, aninflammatory condition, a respiratory condition, organ transplantrejection, or a metabolic disease mediated by or associated with one ormore targets selected from Table 1 or Table 2.

The incorporation of a drug into the inventive fusion proteins providesenhanced compositions that can result in the cure, mitigation,treatment, or prevention of diseases, disorders or conditions in man orother animals. The drug conjugates as represented by formula V orformula VI or formula VII or formula VIII of the present invention areeffective for the usual purposes for which the corresponding drugs areeffective. In one embodiment, the BFP-D compositions can have superiorefficacy compared to the unconjugated drug because of the ability,inherent in the target moiety, to transport the drug to the desiredcells where it is of particular benefit. Exemplary embodiments of theforegoing and representative data are provided in the Examples, below.In another embodiment, the BFP-D and XTEN-drug compositions can havesuperior efficacy compared to the unconjugated drug because of enhancedterminal half-life conferred by the XTEN carrier. In another embodiment,the invention provides BFP-D and XTEN-drug compositions that can havesuperior efficacy, an enhanced pharmacologic response, and/or reducedtoxicity compared to the unconjugated drug because of the differentialcompartmentalization of the composition compared to the unconjugateddrug; e.g., lack of penetration across the blood-brain barrier, nervebarriers, or cytoplasmic barriers of non-targeted cells. Exemplaryembodiments of the foregoing and representative data are provided in theExamples, below. In a particular advantage of the inventivecompositions, such enhanced properties permit lower-dose pharmaceuticalformulations or treatment methods using a reduced dosage or doseregimen, both because of targeted delivery to tissues and cells andbecause of enhanced pharmacokinetic properties, resulting in a superiortherapeutic index; i.e., improved efficacy with reduced toxicity. Theinvention provides for methods of using the conjugate compositions intherapeutic and diagnostic methods, for example for tumor targetingtherapeutics having an altered rate of uptake or tissue diffusion ascompared with the active drug alone.

In one embodiment, the method comprises administering atherapeutically-effective amount of a pharmaceutical compositioncomprising a BFP-D comprising one or more targeting moieties linked toone or more XTEN sequence(s) and at least one pharmaceuticallyacceptable carrier to a subject in need thereof that results in animprovement in at least one parameter, physiologic condition, orclinical outcome mediated by the targeting moiety component(s). Inanother embodiment, the method comprises administering atherapeutically-effective amount of a pharmaceutical compositioncomprising a drug, such as but not limited to a drug selected from Table9, linked to an XTEN sequence and at least one pharmaceuticallyacceptable carrier to a subject in need thereof that results in animprovement in at least one parameter, physiologic condition, orclinical outcome mediated by the targeting moiety component(s). Themethods contemplate administration of the pharmaceutical composition byany route appropriate for the disease, disorder or condition beingtreated, including subcutaneously, intramuscularly, intravitreally, orintravenously.

The methods of the invention may include administration of consecutivedoses of a therapeutically effective amount of the pharmaceuticalcomposition for a period of time sufficient to achieve and/or maintainthe desired parameter or clinical effect, and such consecutive doses ofa therapeutically effective amount establishes the therapeuticallyeffective dose regimen for the pharmaceutical composition; i.e., theschedule for consecutively administered doses, wherein the doses aregiven in therapeutically effective amounts to result in a sustainedbeneficial effect on any clinical sign or symptom, aspect, measuredparameter or characteristic of a metabolic disease state or condition,including, but not limited to, those described herein.

A therapeutically effective amount of the pharmaceutical composition mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the binding fusion protein are outweighed by thetherapeutically beneficial effects. A prophylactically effective amountrefers to an amount of pharmaceutical composition required for theperiod of time necessary to achieve the desired prophylactic result.

For the inventive methods, longer acting BFP-D or XTEN-drug compositionsor pharmaceutical compositions comprising the BFP-D or XTEN-drugcompositions are preferred, so as to improve patient convenience, toincrease the interval between doses and to reduce the amount of drugrequired to achieve a sustained effect. In one embodiment, a method oftreatment comprises administration of a therapeutically effective doseof a BFP-D or an XTEN-drug to a subject in need thereof that results ina gain in time spent within a therapeutic window established for thetargeting moiety or drug components of the pharmaceutical compositioncompared to the corresponding drug component not linked to the fusionprotein and administered at a comparable dose to a subject. In somecases, the gain in time spent within the therapeutic window is at leastabout three-fold, or at least about four-fold, or at least aboutfive-fold, or at least about six-fold, or at least about eight-fold, orat least about 10-fold, or at least about 20-fold, or at least about40-fold compared to the corresponding targeting moiety or drugcomponents not linked to the fusion protein and administered at acomparable dose to a subject. The methods further provide thatadministration of multiple consecutive doses of a pharmaceuticalcomposition administered using a therapeutically effective dose regimento a subject in need thereof can result in a gain in time betweenconsecutive C_(max) peaks and/or C_(min) troughs for blood levels of thecomposition compared to the corresponding targeting moiety or drugcomponents not linked to the fusion protein. In the foregoingembodiment, the gain in time spent between consecutive C_(max) peaksand/or C_(min) troughs can be at least about three-fold, or at leastabout four-fold, or at least about five-fold, or at least aboutsix-fold, or at least about eight-fold, or at least about 10-fold, or atleast about 20-fold, or at least about 40-fold compared to thecorresponding drug component(s) not linked to the fusion protein andadministered using a comparable dose regimen established for that drug.In the embodiments hereinabove described in this paragraph theadministration of the fusion protein or pharmaceutical composition canresult in an improvement in at least one parameter known to be usefulfor assessing the subject diseases, conditions or disorders) using alower unit dose in moles of fusion protein compared to the correspondingtargeting moiety component(s) or the drug component(s) not linked to thefusion protein and administered at a comparable unit dose or doseregimen to a subject.

In one embodiment, the administration of a BFP-D or XTEN-drugpharmaceutical composition can result in an improvement in one of theclinical, biochemical or physiologic parameters that is greater thanthat achieved by administration of the targeting moiety or drugcomponents not linked to XTEN, determined using the same assay or basedon a measured clinical parameter. In another embodiment, administrationof the BFP-D or XTEN-drug pharmaceutical composition can result inimprovement two or more clinical or metabolic-related parameters, eachmediated by one of the different targeting moieties that collectivelyresult in an enhanced effect compared the targeting moiety component notlinked to XTEN, determined using the same assays or based on measuredclinical parameters. In another embodiment, administration of thebinding fusion protein or pharmaceutical composition can result inactivity in one or more of the clinical or biochemical or physiologicparameters that is of longer duration than the activity of one of thesingle targeting moiety or drug components not linked to XTEN,determined using that same assay or based on a measured clinicalparameter.

The subject BFP-D or XTEN-drug conjugate compositions may be useful inthe treatment of various diseases, disorders and conditions. In oneembodiment, the disease is cancer, including carcinomas/tumors,melanomas, sarcomas, leukemias and lymphomas, and gliomas. Exemplaryconditions or hyperproliferative disorders include benign or malignanttumors, neuronal, glial, astrocytal, hypothalamic, glandular,macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenicand immunologic, including autoimmune, disorders. One or morecompositions of the invention may be used in a combination with anothertreatment or drug to treat a patient. As used herein, a “patient” refersto any mammal, including a human, and may be afflicted with any disease,disorder or condition that may be effectively treated with a therapeuticantibody or a drug, including diseases, disorders or conditionsassociated with the targets of Tables 1 or 2 or diseases, disorders orconditions conventionally treated with the drugs of Table 9. Treatmentwith a conjugate may be a primary treatment for a disease or a disorder,or it may be adjuvant therapy for a disease or disorder. Furthermore,the treatment may be of an existing disease or may be prophylactic.Appropriate dosages and dose schedules for the inventive compositionsmay generally be determined using experimental models and/or clinicaltrials. The use of the minimum dosage that is sufficient to provideeffective therapy may be preferred in some circumstances, such as whenthe drug is associated with harmful side effects. Patients may generallybe monitored for therapeutic or prophylactic effectiveness using assayssuitable for the condition being treated or prevented, which will befamiliar to those having ordinary skill in the art. Such methods willpermit the establishment of the therapeutic window as well as themaximum tolerated dose for the compositions of the invention.

The XTEN-drug compositions and/or the binding fusion protein-drugcompositions and pharmaceutical compositions comprising the BFP-D orXTEN-D can be administered by routes and by methods and dosing schedulesappropriate for the given disease, disorder or condition. In certainembodiments, the XTEN-drug compositions and/or the binding fusionprotein-drug compositions of the invention are administered as an IVinfusion. In other embodiments, the compositions are administeredsubcutaneously or intramuscularly. In yet other embodiments, thecompositions are administered orally. In the case of intravenousinfusion, administration may last for any appropriate time period, whichis readily determinable and assessable by one of ordinary skill in theart. For example, infusions may last for from about one to about 24hours, although shorter or longer infusion times all fall within thescope of the invention. In certain embodiments, infusions areadministered daily, weekly, every two weeks, every 21 days, or monthly.Appropriate time periods are known by one of skill in the art, and maybe determined based upon a variety of factors, including the type oftherapy or drug being used in combination with the XTEN-drug conjugate.Clinicians of ordinary skill in the art of medicine will know that thedosage that is administered to a patient will vary according to the age,weight and physical condition of the patient, the route ofadministration, the specific disease being treated, the stage of diseaseand the like. For any particular subject, the specific dosage regimens(both dosage and frequency of administration) should be adjusted forthat patient by a skilled practitioner. Examples of different ranges ofdosage and administration schedules are provided in U.S. Pat. No.5,670,537.

Targeted XTEN-drug conjugate compositions useful in the treatment ofcancer include, but are not limited to, compositions comprising one ormore targeting moieties against cell surface receptors andtumor-associated antigens (TAA). Tumor-associated antigens are known inthe art, and can be prepared for use in generating targeting moietiesusing methods and information that are well known in the art.Non-limiting examples of tumor-associated polypeptides are containedwithin Table 2. In preferred embodiments, the targets to which targetingmoieties would be directed could include receptors specificallyexpressed on the surface of one or more particular type(s) of cancercell as compared to on one or more normal non-cancerous cells. Often,such tumor-associated polypeptides are more abundantly expressed on thesurface of the cancer cells as compared to on the surface of thenon-cancerous cells; e.g., HER2 in certain breast cancers. Theidentification of such tumor-associated cell surface antigenpolypeptides has given rise to the ability to specifically target cancercells for destruction via antibody-based therapies.

Disorders Mediated by VEGF-Expressing Cells

A BFP-D composition that comprises a targeting moiety derived from ananti-VEGF antibody or fragment can be advantageously utilized in amethod of treating a VEGF-mediated disease or disorder, such asneovascular disorders. In one embodiment, the invention provides amethod of treating a solid tumor disorder in a human patient comprisingadministering to the patient an effective amount of a BFP-D orpharmaceutical composition wherein at least one targeting moiety in thebinding fusion protein comprises an antigen binding site that binds tohuman VEGF wherein the binding can inhibit vascularization of the tumor,and the BFP-D further comprises a conjugated cytotoxic drug, such as adrug selected from Table 9, for treating a solid tumor. In yet anotherembodiment, the solid tumor disorder in the foregoing method is selectedfrom the group consisting of breast carcinomas, lung carcinomas, gastriccarcinomas, esophageal carcinomas, colorectal carcinomas, livercarcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervicalcarcinomas, endometrial carcinoma, endometrial hyperplasia,endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer,nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi'ssarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma,hemangioblastoma, pancreas carcinomas, retinoblastoma, astrocytoma,glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma,neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas,urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, renal cellcarcinoma, prostate carcinoma, abnormal vascular proliferationassociated with phakomatoses, edema (such as that associated with braintumors), and Meigs' syndrome.

In still another embodiment, the invention provides a method of treatingan intraocular neovascular disorder in a human patient comprisingadministering to the patient a therapeutically effective amount of aBFP-D or pharmaceutical composition comprising the BFP-D wherein atleast one targeting moiety comprises an antigen binding site that bindsto human VEGF and the BFP-D further comprises a conjugated cytotoxicdrug, such as a drug selected from Table 9. In a further embodiment, theintraocular neovascular disorder is selected from the group consistingof diabetic and other proliferative retinopathies including retinopathyof prematurity, retrolental fibroplasia, neovascular glaucoma, andage-related macular degeneration.

Disorders Mediated by HER2-Expressing Cells

In another embodiment, the binding fusion protein is used to treatdisorders mediated by HER2-expressing cells. The invention provides amethod for treating a human disease mediated by HER2-expressing cellswith a binding fusion protein composition that is derived from aparental antibody that binds to HER2. Such compositions haveprophylactic and therapeutic applications in a broad spectrum ofHER2-expressing cell-mediated disorders, including pathologies supportedby the proliferation of cells expressing HER2, such as cancerscharacterized by over-expression of HER2, in a manner similar to theapplication of full length anti-Her2 antibodies in the treatment of suchdisease indications that is known in the art, which treatmentindications include HER2-overexpressing breast, ovarian and lungcancers.

In one embodiment, the invention provides a method of treating aHER2-expressing cell mediated disorder in a human patient comprisingadministering to the patient a therapeutically effective amount of aBFP-D or pharmaceutical composition comprising a BFP-D wherein at leastone targeting moiety in the binding fusion protein comprises an antigenbinding site that binds to HER2, and the BFP-D further comprises aconjugated cytotoxic drug, such as a drug selected from Table 9. Thedisorder can be a HER2-expressing cell proliferative disorder, includinga benign or malignant tumor characterized by the over-expression of theErbB2 receptor, e.g. a cancer, such as, breast cancer, squamous cellcancer, small-cell lung cancer, non-small cell lung cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, coloncancer, colorectal cancer, endometrial carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer. In addition, the invention contemplates the use of the foregoingconjugate in place of full-length anti-Her2 antibody in the treatment ofHER2-overexpressing cancers as described in U.S. Pat. No. 5,725,856.

In one non-limiting exemplary method, the invention provides a method ofinhibiting growth of tumor cells by administering to a patient atherapeutically effective amount of anti-HER2 BFP-D composition capableof inhibiting the HER2 receptor function by the targeting moietycomponent and/or inhibiting or killing the HER2 cell by the drugcomponent, such as, but not limited to epaclitaxel, paclitaxel,docetaxel, doxetaxel, irinotecan, pemetrexed, chloranbucil, orgemcitabine, or a suitable cytotoxic drug selected from Table 9.

A further embodiment of the invention relates to administering atherapeutically effective amount of anti-HER2 composition capable ofinhibiting growth factor receptor function. Still another object of theinvention is to provide methods for the treatment and/or prevention oferbB-2 receptor over-expressing tumors comprising the administration ofan anti-tumor effective amount of at least one of the disclosedanti-HER2 BFP-D capable of binding to cancer cells associated by theover-expression of erbB-2 protein. In another embodiment, the inventionprovides a method for the treatment and/or prevention of erbB-2 receptorover-expressing tumors comprising the administration oftherapeutically-effective amounts of anti-Her2 BFP-D comprising a firstand a second anti-Her2 binding moiety, which may be the same or whichmay bind different epitopes of the erbB-2 protein, capable of inhibitingthe HER2 receptor function, and one or more drug molecules selected fromTable 9. Preferably, such combinations of binding moieties and drug willexhibit better cytotoxic activity than would be expected for the sum ofthe cytotoxic activity of the individual antibodies and separatelyadministered drugs at the same or lower overall concentrations of theindividual components.

Disorders Mediated by EGFR-Expressing Cells

In one embodiment, the invention provides a method for treating a humandisease mediated by EGFR-expressing cells with a BFP-D that is derivedfrom a parental antibody that binds to human EGFR (a.k.a., ErbB-1 orHer1) and further comprises a drug selected from Table 9. Such BFP-D canhave prophylactic and therapeutic applications in a broad spectrum ofEGFR-expressing cell-mediated disorders, including pathologies supportedby the proliferation of cells expressing EGFR, such as cancerscharacterized by over-expression of EGFR, including cancers of thebreast, ovary, head and neck, brain, bladder, pancreas, and lung.

In one embodiment, the invention provides a method of treating a cellproliferation disorder in a human patient characterized byover-expression of EGFR comprising administering to the patient atherapeutically effective amount of a BFP-D wherein at least onetargeting moiety in the binding fusion protein comprises an antigenbinding site that binds to human EGFR and further comprises a cytotoxicdrug known to be effective against EGFR-bearing cells. The disorder canbe a benign or malignant tumor characterized by the over-expression ofthe EGFR, e.g. a cancer, such as, breast cancer, squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, colon cancer, colorectalcancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer,liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer.

Disorders Mediated by CD20-Expressing Cells

In another embodiment, the invention provides a method of treatingdisorders mediated by CD20-expressing cells. The invention provides amethod for treating a human disease mediated by CD20-expressing cellswith a BFP-D composition that is derived from a parental antibody thatbinds to human CD20 and further comprises a cytotoxic drug known to haveactivity against CD20 expressing cells, such as a drug selected fromTable 9. Such compositions have prophylactic and therapeuticapplications in a broad spectrum of CD20-expressing cell-mediateddisorders, including pathologies supported by the proliferation ofCD20-expressing cells, such as cancers of CD20-expressing cells, in amanner similar to the application of full length anti-CD20 antibodies inthe treatment of such disease indications known in the art, whichtreatment indications include B-lymphocytic lymphomas, as described inU.S. Pat. No. 6,682,734.

Disorders Mediated by CD18-Expressing Cells

In another embodiment, the invention provides a method of treatingdisorders mediated by CD18-expressing cells. The invention provides amethod for treating a human disease mediated by CD18-expressing cellswith a BFP-D composition that is derived from a parental antibody thatbinds to human CD18 and further comprises a cytotoxic drug known to haveactivity against CD18 expressing cells, such as a drug selected fromTable 9. Such compositions have prophylactic and therapeuticapplications in a broad spectrum of CD18-expressing cell-mediateddisorders, including pathologies supported by leukocyte adhesion, in amanner similar to the application of full length anti-CD18 antibodies inthe treatment of such disease indications known in the art, whichtreatment indications include acute myocardial infarction and stroke. Inone embodiment, the invention provides a method of treating a disorderin a human patient mediated by a CD18-expressing cell, comprisingadministering to the patient a therapeutically effective amount of aBFP-D wherein at least one targeting moiety in the conjugate comprisesan antigen binding site that binds to human CD18, and the BFP-D furthercomprises a conjugated drug known to have beneficial effects in thetreatment of myocardial infarction and stroke, such as a drug selectedfrom Table 9. In another embodiment, the CD18-expressing cell-mediateddisorder is an inflammatory disorder, such as an ischemic reperfusiondisorder, including acute myocardial infarction and stroke. In addition,the invention contemplates the use of the foregoing binding fusionprotein in place of full-length anti-CD18 antibody in the treatment ofstroke as described in PCT Publication WO 97/26912.

In another embodiment, the invention provides a method of treating aLFA-1-mediated disorder in a human, comprising administering to thepatient a therapeutically effective amount of a binding fusion proteinwherein at least one targeting moiety in the conjugate comprises atargeting moiety that binds to human CD18, and the BFP-D furthercomprises a conjugated immunosuppressive drug, such as a drug selectedfrom Table 9. In addition, the invention contemplates the use of theforegoing binding fusion protein in place of full-length anti-CD18antibody in the treatment of an LFA-1-mediated disorder, such aspsoriasis and graft rejection, in a human patient as described in U.S.Pat. No. 5,622,700.

Disorders Mediated by CD11a-Expressing Cells

In another embodiment, the invention provides a method of treatingdisorders mediated by CD11a-expressing cells. In one embodiment, theinvention provides a method for treating a human disease mediated by aCD11a-expressing cell with a binding fusion protein composition that isderived from a parental antibody that binds to human CD11a and furthercomprises an immunosuppressive or cytotoxic drug known to have activityagainst CD11a expressing cells. Such compositions have prophylactic andtherapeutic applications in a broad spectrum of CD11a-expressingcell-mediated disorders, including pathologies supported by leukocyteadhesion, in a manner similar to the application of full lengthanti-CD11a antibodies in the treatment of such disease indications knownin the art, which treatment indications include psoriasis, asthma, graftrejection, and multiple sclerosis. In another embodiment, the inventionprovides a method of treating a LFA-1-mediated disorder in a human,comprising administering to the patient a therapeutically effectiveamount of a binding fusion protein wherein at least one targeting moietyin the conjugate comprises an antigen binding site that binds to humanCD11a. In addition, the invention contemplates the use of the foregoingbinding fusion protein in place of full-length anti-CD11a antibody inthe treatment of an LFA-1-mediated disorder, such as psoriasis and graftrejection, in a human patient as described in U.S. Pat. No. 5,622,700.In another aspect, the invention contemplates the use of the foregoingbinding fusion proteins in place of full-length anti-CD11a antibody inthe treatment of LFA-1-mediated disorders in a human patient asdescribed in U.S. Pat. No. 6,037,454.

Disorders Mediated by CD3-Expressing Cells

In one embodiment, the invention provides a method for treating a humandisease or disorder mediated by CD3-expressing cells with a bindingfusion protein that is derived from a parental antibody that binds tohuman CD3 and further comprises a cytotoxic or immunosuppressive drugknown to have activity against CD3 expressing cells, such as a drugselected from Table 9. Such binding fusion proteins can haveprophylactic and therapeutic applications in a broad spectrum ofCD3-expressing cell-mediated disorders, including conditions associatedwith the proliferation or activation of cells expressing CD3, such asimmune disorders mediated by T-lymphocytes and graft rejection intransplant recipients. The use of anti-CD3 antibodies to treat diseasesand disorders has been described, for example, in U.S. Pat. No.4,515,893. In another aspect, the invention contemplates the use of theforegoing binding fusion protein in place of full length anti-human CD3antibody in the treatment of acute allograft rejection in kidneytransplant recipients as described for ORTHOCLONE OKT3 muromonab-CD3 inPhysician's Desk Reference, 52^(nd) Edition (1998), pp. 1971-1974.

Disorders Mediated by TAC-Expressing Cells

In one embodiment, the invention provides a method for treating a humandisease mediated by interleukin-2 receptor α-chain (TAC)-expressingcells with a binding fusion protein that is derived from a parentalantibody that binds to human TAC and further comprises a cytotoxic drugknown to have activity against TAC expressing cells. Such binding fusionproteins can have prophylactic and therapeutic applications in a broadspectrum of TAC-expressing cell-mediated disorders, including conditionscreated by the proliferation or activation of cells expressing TAC andimmune disorders mediated by T-lymphocytes or B-lymphocytes, includinggraft rejection in transplant recipients.

In one embodiment, the invention provides a method of treating adisorder in a human patient mediated by a TAC-expressing cell,comprising administering to the patient a therapeutically effectiveamount of a binding fusion protein wherein at least one targeting moietyin the binding fusion protein comprises an antigen binding site thatbinds to human TAC, and the BFP-D further comprises a conjugatedimmunosuppressive or cytotoxic drug, such as a drug selected from Table9. In another embodiment, the TAC-expressing cell-mediated disorder ischaracterized by the activation or proliferation of T-lymphocytes orB-lymphocytes, including immune disorders such as graft rejection intransplant recipients, graft-versus-host disease (GHVD), graft rejectionin transplant recipients, such as acute graft rejection in renaltransplant recipients, and autoimmune diseases such as Type I diabetes,multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus,and myasthenia gravis. The use of antibodies to treat disorders mediatedby interleukin-2 receptor α-chain with antibodies has been described inU.S. Pat. No. 5,693,761.

TNF-α-Mediated Disorders

In one embodiment, the invention provides a method for treating aTNF-α-mediated disease with a binding fusion protein that is derivedfrom a parental antibody that binds to human TNF-α and further comprisesa cytotoxic or anti-inflammatory drug, such as a drug selected fromTable 9. Such binding fusion proteins can have prophylactic andtherapeutic applications in a broad spectrum of TNF-α-mediateddisorders, including inflammatory disorders and immune disorders, in amanner similar to the application of full-length anti-human TNF-αantibodies in the treatment of such disease indications such as Crohn'sdisease, inflammatory bowel disease, and rheumatoid arthritis.

In one embodiment, the invention provides a method of treating aninflammatory disorder in a human patient comprising administering to thepatient a therapeutically effective amount of a binding fusion proteinwherein at least one targeting moiety in the binding fusion proteincomprises an antigen binding site that binds to human TNF-α. In anotherembodiment, the inflammatory disorder is Crohn's disease. In yet anotherembodiment, the inflammatory disorder is inflammatory bowel disease. Instill another embodiment, the inflammatory disorder is rheumatoidarthritis. The use of antibodies that bind to human TNF-α in thetreatment of inflammatory conditions have been described, for example,in U.S. Pat. Nos. 5,672,347, 5,656,272, and 5,698,195.

Tissue Factor-Mediated Disorders

In one embodiment, the invention provides a method for treating a tissuefactor-mediated disease with a binding fusion protein derived from aparental antibody that binds to human tissue factor, and the BFP-Dfurther comprises a conjugated anticoagulant or antithrombosis drug,such as a drug selected from Table 9. Such binding fusion proteins canhave prophylactic and therapeutic applications in a broad spectrum oftissue factor-mediated disorders, including pathologies supported byblood coagulation and in the treatment of such disease indications asdeep vein thrombosis, arterial thrombosis, atherosclerosis, vascularstenosis, myocardial ischemic diseases including acute myocardialinfarction, reocclusion following angioplasty or atherectomy orthrombolytic treatment for acute myocardial infarction, angina, cerebralischemic diseases including stroke, venous thrombophlebitis, andpulmonary embolism. In one embodiment, the invention provides a methodof treating a tissue factor-mediated disease or disorder (such as theforegoing) in a human patient comprising administering to the patient atherapeutically effective amount of a binding fusion protein wherein atleast one targeting moiety in the binding fusion protein comprises anantigen binding site that binds to human tissue factor.

III). The DNA Sequences of the Invention

The present invention provides isolated polynucleic acids encoding XTENand binding fusion protein chimeric polypeptides and sequencescomplementary to polynucleic acid molecules encoding XTEN and bindingfusion protein chimeric polypeptides, including homologous variants. Inanother aspect, the invention encompasses methods to produce polynucleicacids encoding XTEN and binding fusion protein chimeric polypeptides andsequences complementary to polynucleic acid molecules encoding bindingfusion protein chimeric polypeptides, including homologous variants. Ingeneral, and as illustrated in FIGS. 7-9, the methods of producing apolynucleotide sequence coding for an XTEN or a binding fusion proteinand expressing the resulting gene product include assembling nucleotidesencoding targeting moieties and XTEN (and any linker sequences, if any),linking the components in frame, incorporating the encoding gene into anappropriate expression vector, transforming an appropriate host cellwith the expression vector, and causing the fusion protein to beexpressed in the transformed host cell, thereby producing thebiologically-active binding fusion protein. Standard recombinanttechniques in molecular biology can be used to make the polynucleotidesand expression vectors of the present invention.

In accordance with the invention, nucleic acid sequences that encodeXTEN and binding fusion proteins may be used to generate recombinant DNAmolecules that direct the expression of XTEN and binding fusion proteinsin appropriate host cells. Several cloning strategies are envisioned tobe suitable for performing the present invention, many of which can beused to generate a construct that comprises a gene coding for a bindingfusion protein composition of the present invention, or its complement.In one embodiment, the cloning strategy would be used to create a genethat encodes an XTEN polypeptide. In another embodiment, the cloningstrategy would be used to create a gene that encodes a monomeric bindingfusion protein that comprises at least a first targeting moiety and atleast a first XTEN polypeptide, or its complement. In anotherembodiment, the cloning strategy would be used to create a gene thatencodes a monomeric binding fusion protein that comprises a first and asecond targeting moiety and at least a first XTEN, or its complement. Inanother embodiment, the cloning strategy would be used to create a genethat encodes a monomeric binding fusion protein that comprises at leasta first and a second targeting moiety, a linker, and at least a firstXTEN, or its complement. In the foregoing embodiments, the gene would beused in a suitable expression vector to transform a host cell forexpression of the fusion protein.

In designing a desired XTEN sequences, it was discovered that thenon-repetitive nature of the XTEN of the inventive compositions can beachieved despite use of a “building block” molecular approach in thecreation of the XTEN-encoding sequences. This was achieved by the use ofa library of polynucleotides encoding sequence motifs that are thenmultimerized to create the genes encoding the XTEN sequences (see FIGS.7 and 8). Thus, while the expressed XTEN may consist of multiple unitsof as few as four different sequence motifs, because the motifsthemselves consist of non-repetitive amino acid sequences, the overallXTEN sequence is rendered non-repetitive. Accordingly, in oneembodiment, the XTEN-encoding polynucleotides comprise multiplepolynucleotides that encode non-repetitive sequences, or motifs,operably linked in frame and in which the resulting expressed XTEN aminoacid sequences are non-repetitive.

In one approach, a construct is first prepared containing the DNAsequence corresponding to binding fusion protein. DNA encoding thetargeting moiety of the compositions may be obtained from a cDNA libraryprepared using standard methods from tissue or isolated cells believedto possess targeting moiety mRNA and to express it at a detectablelevel. If necessary, the coding sequence can be obtained usingconventional primer extension procedures as described in Sambrook, etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA. Accordingly, DNAcan be conveniently obtained from a cDNA library prepared from suchsources. The target moiety encoding gene(s) may also be obtained from agenomic library or created by standard synthetic procedures known in theart (e.g., automated nucleic acid synthesis) using DNA sequencesobtained from publicly available databases, patents, or literaturereferences. Such procedures are well known in the art and well describedin the scientific and patent literature. For example, sequences can beobtained from Chemical Abstracts Services (CAS) Registry Numbers(published by the American Chemical Society) and/or GenBank AccessionNumbers (e.g., Locus ID, NM_XXXXX, NP_XXXXX, and XP_XXXXX) Model Proteinidentifiers available through the National Center for BiotechnologyInformation (NCBI) webpage, available on the world wide web atncbi.nlm.nih.gov that correspond to entries in the CAS Registry orGenBank database that contain an amino acid sequence of the targetingmoiety (e.g., an antibody) or of a fragment or variant of the targetingmoiety. For such sequence identifiers provided herein, the summary pagesassociated with each of these CAS and GenBank and GenSeq AccessionNumbers as well as the cited journal publications (e.g., PubMed IDnumber (PMID)) are each incorporated by reference in their entireties,particularly with respect to the amino acid sequences described therein.In one embodiment, the binding fusion protein encoding gene encodes aprotein from any one of Tables 25, 38 or 39, or a fragment or variantthereof.

A gene or polynucleotide encoding the targeting moiety portion of thesubject binding fusion protein, in the case of an expressed fusionprotein that will comprise a single targeting moiety, can be then becloned into a construct, which can be a plasmid or other vector undercontrol of appropriate transcription and translation sequences for highlevel protein expression in a biological system. In a later step, asecond gene or polynucleotide coding for the XTEN is genetically fusedto the nucleotides encoding the N- and/or C-terminus of the targetingmoiety gene by cloning it into the construct adjacent and in frame withthe gene(s) coding for the targeting moiety. This second step can occurthrough a ligation or multimerization step. In the foregoing embodimentshereinabove described in this paragraph, it is to be understood that thegene constructs that are created can alternatively be the complement ofthe respective genes that encode the respective fusion proteins. Inaddition, for binding fusion proteins comprising two or more targetingmoieties and linkers, the gene or polynucleotides coding for thesecomponents would be cloned into the construct adjacent to and in framerelative to the other components described above, depending on thedesired final configuration of the fusion protein. In a particularaspect of the foregoing, it was discovered that use of alternativeencoding sequences for multivalent (e.g., two or more) targetingmoieties reduces the risk of homologous recombination during expression.Accordingly, for binding fusion proteins that have repeat bindingdomains or multivalent targeting moieties with the same or very similarsequences, the invention provides encoding polynucleotides for therespective domains that have different DNA sequences. In a non-limitingexample of the foregoing, the invention provides a binding fusionprotein with dimeric Ig-like targeting moieties wherein the codons forcertain amino acids for the encoding gene for each targeting moiety arevaried, wherein the incidence of recombination during expression in atransformed host is reduced compared to a comparable host transformedwith targeting genes which are identical.

The gene encoding for the XTEN can be made in one or more steps, eitherfully synthetically or by synthesis combined with enzymatic processes,such as restriction enzyme-mediated cloning, PCR and overlap extension.XTEN polypeptides can be constructed such that the XTEN-encoding genehas low repetitiveness. Genes encoding XTEN with non-repetitivesequences can be assembled from oligonucleotides using standardtechniques of gene synthesis. The gene design can be performed usingalgorithms that optimize codon usage and amino acid composition. In onemethod of the invention, a library of relatively short XTEN-encodingpolynucleotide constructs is created and then assembled, as illustratedin FIGS. 7 and 8. This can be a pure codon library such that eachlibrary member has the same amino acid sequence but many differentcoding sequences are possible. Such libraries can be assembled frompartially randomized oligonucleotides and used to generate largelibraries of XTEN segments comprising the sequence motifs. Therandomization scheme can be optimized to control amino acid choices foreach position as well as codon usage.

Polynucleotide Libraries

In another aspect, the invention provides libraries of polynucleotidesthat encode XTEN sequences that can be used to assemble genes thatencode XTEN of a desired length and sequence, which are useful for thecreation of genes encoding binding fusion proteins.

In certain embodiments, the XTEN-encoding library constructs comprisepolynucleotides that encode polypeptide segments of a fixed length. Asan initial step, a library of oligonucleotides that encode motifs of9-14 amino acid residues can be assembled. In a preferred embodiment,libraries of oligonucleotides that encode motifs of 12 amino acids areassembled.

The XTEN-encoding sequence segments can be dimerized or multimerizedinto longer encoding sequences. Dimerization or multimerization can beperformed by ligation, overlap extension, PCR assembly or similarcloning techniques known in the art. This process of can be repeatedmultiple times until the resulting XTEN-encoding sequences have reachedthe organization of sequence and desired length, providing theXTEN-encoding genes. As will be appreciated, a library ofpolynucleotides that encodes 12 amino acids can be dimerized into alibrary of polynucleotides that encode 36 amino acids. In turn, thelibrary of polynucleotides that encode 36 amino acids can be seriallydimerized into a library containing successively longer lengths ofpolynucleotides that encode XTEN sequences. In some embodiments,libraries can be assembled of polynucleotides that encode amino acidsthat are limited to specific sequence XTEN families; e.g., AD, AE, AF,AG, AM, AQ, BC, or BD sequences of Table 3. In other embodiments,libraries can comprises sequences that encode two or more of the motiffamily sequences from Table 3. The names and sequences ofrepresentative, non-limiting polynucleotide sequences of libraries thatencode 36mers are presented in Tables 11-14, and the methods used tocreate them are described more fully in the Examples. The libraries canbe used, in turn, for serial dimerization or ligation to achievepolynucleotide sequence libraries that encode XTEN sequences, forexample, of 72, 144, 288, 576, 864, 912, 923, 1296 amino acids, or up toa total length of about 3000 amino acids, as well as intermediatelengths. In one embodiment, the polynucleotide library sequences mayalso include additional bases used as “sequencing islands,” describedmore fully below.

FIG. 8 is a schematic flowchart of representative, non-limiting steps inthe assembly of a XTEN polynucleotide construct and a binding fusionprotein polynucleotide construct utilized in the XTEN, binding fusionprotein and BFP-D embodiments of the invention. Individualoligonucleotides 501 can be annealed into sequence motifs 502 such as a12 amino acid motif (“12-mer”), which is subsequently ligated with anoligo containing BbsI, and KpnI restriction sites 503. Additionalsequence motifs from a library are annealed to the 12-mer until thedesired length of the XTEN gene 504 is achieved. The XTEN gene is clonedinto a stuffer vector. The vector can optionally encode a Flag sequence506 followed by a stuffer sequence that is flanked by BsaI, BbsI, andKpnI sites 507 and, in this case, a single targeting moiety gene(encoding anti-Her2 in this example) 508, resulting in the gene encodinga binding fusion protein comprising a single targeting moiety 500. Anon-exhaustive list of the XTEN names and sequences for polynucleotidesencoding XTEN and precursor sequences is provided in Table 10.

TABLE 10 DNA sequences of XTEN and precursor sequences SEQ XTEN ID NameDNA Sequence NO: AE144GGTAGCGAACCGGCAACTTCCGGCTCTGAAACCCCAGGTACTTCTGAAAGCGCTAC 202TCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCTGGCTCTGAAACCCCAGGTAGCCCGGCAGGCTCTCCGACTTCCACCGAGGAAGGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAAACTCCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACTCCAGGTACCTCTACCGAACCTTCCGAAGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA AF144GGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGA 203ATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAGGTTCTACTAGCTCTACCGCAGAATCTCCGGGTCCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTACTCCGGAAAGCGGCTCCGCATCTCCAGGTTCTACTAGCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCACCA AE288GGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTC 204CGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA AE576GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTAC 205TCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA AF576GGTTCTACTAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCCACTAGCTCTACCGCA 206GAATCTCCGGGCCCAGGTTCTACTAGCGAATCCCCTTCTGGTACCGCTCCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCAGAATCTCCTGGCCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTTCCACTAGCTCTACCGCTGAATCTCCGGGTCCAGGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTACCCCGGAAAGCGGCTCTGCTTCTCCAGGTACTTCTACCCCGGAAAGCGGCTCCGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCTACCAGCGAATCTCCTTCTGGTACTGCACCAGGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTTCCACTAGCTCTACTGCTGAATCTCCTGGCCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCA AM875GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACTTC 207CGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCACCAGGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTAGCCCGTCTGCATCTACCGGTACCGGCCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCAGGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCAGGTTCCACCAGCTCTACTGCAGAATCTCCGGGCCCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAGGTAGCGAACCTGCAACCTCCGGCTCTGAAACCCCAGGTACTTCTACTGAACCTTCTGAGGGCAGCGCACCAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTAGCGAACCTGCTACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCAGGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA AE864GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTAC 208TCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA AF864GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGA 209ATCTTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTTCTACTAGCGAATCTCCGTCTGGCACTGCTCCAGGTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCCCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCAGGTACCTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCTCCGAGCGGTGAATCTTCTACCGCTCCAGGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAGGTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAGGTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCAGGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTCCXXXXXXXXXXXXTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAXXXXXXXXTAGCGAATCTCCTTCTGGTACCGCTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCTACCAGCGAATCTCCTTCTGGTACTGCACCAGGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCTACCAGCGAATCTCCTTCTGGTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCAGGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAGGTACTTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTACCCCGGAAAGCGGCTCTGCTTCTCCAGGTACTTCTACCCCGGAAAGCGGCTCCGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTTCCACTAGCTCTACCGCTGAATCTCCGGGTCCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAGGTACTTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTTCTAGCCCTTCTGCTTCCACCGGTACCGGCCCAGGTAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAGGTAGCTCTACTCCGTCTGGTGC AACCGGCTCCCCAXXXX was inserted in two areas where no sequenceinformation is available. AG864GGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTTCTAGCCCGTCTGCTTCT 210ACTGGTACTGGTCCAGGTTCTAGCCCTTCTGCTTCCACTGGTACTGGTCCAGGTACCCCGGGTAGCGGTACCGCTTCTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAGGTTCTAACCCTTCTGCATCCACCGGTACCGGCCCAGGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAGGTACCCCGGGCAGCGGTACCGCATCTTCTTCTCCAGGTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCAGGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAGGTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTACCCCGGGTAGCGGTACCGCTTCTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAGGTTCTAACCCTTCTGCATCCACCGGTACCGGCCCAGGTTCTAGCCCTTCTGCTTCCACCGGTACTGGCCCAGGTAGCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGCTCTACTCCTTCTGGTGCAACTGGCTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAGGTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCAGGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCAGGTACTCCTGGCAGCGGTACTGCATCTTCCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTACCCCTGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGTTCTCCAGGTACCCCGGGTAGCGGTACCGCATCTTCTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCCCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGGTTCTAGCCCGTCTGCATCTACTGGTACTGGTCCAGGTGCATCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTACTCCTGGTAGCGGTACTGCTTCTTCTTCTCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGTTCTCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCAGGTTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGTGCTTCTCCGGGTACTAGCTCTACTGGTTCTCCAGGTGCATCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCTCCAGGTTCTAGCCCTTCTGCATCTACCGGTACTGGTCCAGGTGCATCCCCTGGTACCAGCTCTACCGGTTCTCCAGGTTCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGGTACCCCTGGCAGCGGTACCGCATCTTCCTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCA AM923ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCATCCCCGGGCAC 211CAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCACCAGGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTAGCCCGTCTGCATCTACCGGTACCGGCCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCAGGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCAGGTTCCACCAGCTCTACTGCAGAATCTCCGGGCCCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAGGTAGCGAACCTGCAACCTCCGGCTCTGAAACCCCAGGTACTTCTACTGAACCTTCTGAGGGCAGCGCACCAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTAGCGAACCTGCTACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCAGGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCG AAGGTAGCGCACCAAE912 ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGCGG 212TACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA AM1296GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACTTC 213CGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCACCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTTCTGAAAGCGCTACTCCGGAATCCGGTCCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTTCTAGCCCTTCTGCTTCCACCGGTACCGGCCCAGGTAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTGCATCCCCGGGTACTAGCTCTACCGGTTCTCCAGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTAGCTCTACTCCTTCTGGTGCTACCGGCTCTCCAGGTGCTTCTCCGGGTACTAGCTCTACCGGTTCTCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTTCTACCAGCGAATCCCCTTCTGGCACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGTTCTACCAGCTCTACCGCAGAATCTCCGGGTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCAGGTACTCCGGGTAGCGGTACCGCTTCTTCCTCTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGA AGGTAGCGCTCCABC864 GGTACTTCCACCGAACCATCCGAACCAGGTAGCGCAGGTACTTCCACCGAACCATC 214CGAACCTGGCAGCGCAGGTAGCGAACCGGCAACCTCTGGTACTGAACCATCAGGTAGCGGCGCATCCGAGCCTACCTCTACTGAACCAGGTAGCGAACCGGCTACCTCCGGTACTGAGCCATCAGGTAGCGAACCGGCAACTTCCGGTACTGAACCATCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGTGCATCTGAGCCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAGCGCAGGTAGCGAACCAGCTACTTCTGGCACTGAACCATCAGGTACTTCTACTGAACCATCCGAACCAGGTAGCGCAGGTAGCGAACCTGCTACCTCTGGTACTGAGCCATCAGGTAGCGAACCGGCTACCTCTGGTACTGAACCATCAGGTACTTCTACCGAACCATCCGAGCCTGGTAGCGCAGGTACTTCTACCGAACCATCCGAGCCAGGCAGCGCAGGTAGCGAACCGGCAACCTCTGGCACTGAGCCATCAGGTAGCGAACCAGCAACTTCTGGTACTGAACCATCAGGTACTAGCGAGCCATCTACTTCCGAACCAGGTGCAGGTAGCGGCGCATCCGAACCTACTTCCACTGAACCAGGTACTAGCGAGCCATCCACCTCTGAACCAGGTGCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGAACCGGCTACCTCTGGTACTGAACCATCAGGTACTTCTACCGAACCATCCGAGCCTGGTAGCGCAGGTACTTCTACCGAACCATCCGAGCCAGGCAGCGCAGGTAGCGGTGCATCCGAGCCGACCTCTACTGAACCAGGTAGCGAACCAGCAACTTCTGGCACTGAGCCATCAGGTAGCGAACCAGCTACCTCTGGTACTGAACCATCAGGTAGCGAACCGGCTACTTCCGGCACTGAACCATCAGGTAGCGAACCAGCAACCTCCGGTACTGAACCATCAGGTACTTCCACTGAACCATCCGAACCGGGTAGCGCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGTGCATCTGAGCCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAGCGCAGGTAGCGAACCTGCAACCTCCGGCACTGAGCCATCAGGTAGCGGCGCATCTGAACCAACCTCTACTGAACCAGGTACTTCCACCGAACCATCTGAGCCAGGCAGCGCAGGTAGCGGCGCATCTGAACCAACCTCTACTGAACCAGGTAGCGAACCAGCAACTTCTGGTACTGAACCATCAGGTAGCGGCGCATCTGAGCCTACTTCCACTGAACCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGTGCATCTGAGCCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAGCGCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGTGCATCTGAGCCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAGCGCAGGTAGCGAACCAGCTACTTCTGGCACTGAACCATCAGGTACTTCTACTGAACCATCCGAACCAGGTAGCGCAGGTAGCGAACCTGCTACCTCTGGTACTGAGCCATCAGGTACTTCTACTGAACCATCCGAGCCGGGTAGCGCAGGTACTTCCACTGAACCATCTGAACCTGGTAGCGCAGGTACTTCCACTGAACCATCCGAACCAGGTAGCGCAGGTACTTCTACTGAACCATCCGAGCCGGGTAGCGCAGGTACTTCCACTGAACCATCTGAACCTGGTAGCGCAGGTACTTCCACTGAACCATCCGAACCAGGTAGCGCAGGTACTAGCGAACCATCCACCTCCGAACCAGGCGCAGGTAGCGGTGCATCTGAACCGACTTCTACTGAACCAGGTACTTCCACTGAACCATCTGAGCCAGGTAGCGCAGGTACTTCCACCGAACCATCCGAACCAGGTAGCGCAGGTACTTCCACCGAACCATCCGAACCTGGCAGCGCAGGTAGCGAACCGGCAACCTCTGGTACTGAACCATCAGGTAGCGGTGCATCCGAGCCGACCTCTACTGAACCAGGTAGCGAACCAGCAACTTCTGGCACTGAGCCATCAGGTAGCGAACCAGCTACCTCTGGTACTGAACCATCAGGTAGCGAACCGGCAACCTCTGGCACTGAGCCATCAGGTAGCGAACCAGCAACTTCTGGTACTGAACCATCAGGTACTAGCGAGCCATCTACTTCCGAACCAGGTGCAGGTAGCGAACCTGCAACCTCCGGCACTGAGCCATCAGGTAGCGGCGCATCTGAACCAACCTCTACTGAACCAGGTACTTCCACCGAACCATCTGAGCCAGGCAGCGCAGGTAGCGAACCTGCAACCTCCGGCACTGAGCCATCAGGTAGCGGCGCATCTGAACCAACCTCTACTGAACCAGGTACTTCCACCGAACCATCTGAGCCAGGCAGCGCA BD864GGTAGCGAAACTGCTACTTCCGGCTCTGAGACTGCAGGTACTAGTGAATCCGCAAC 215TAGCGAATCTGGCGCAGGTAGCACTGCAGGCTCTGAGACTTCCACTGAAGCAGGTACTAGCGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACTGCTACCTCTGGCTCCGAGACTGCAGGTAGCGAAACTGCAACCTCTGGCTCTGAAACTGCAGGTACTTCCACTGAAGCAAGTGAAGGCTCCGCATCAGGTACTTCCACCGAAGCAAGCGAAGGCTCCGCATCAGGTACTAGTGAGTCCGCAACTAGCGAATCCGGTGCAGGTAGCGAAACCGCTACCTCTGGTTCCGAAACTGCAGGTACTTCTACCGAGGCTAGCGAAGGTTCTGCATCAGGTAGCACTGCTGGTTCCGAGACTTCTACTGAAGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCAGGTACTAGCGAGTCCGCTACTAGCGAATCTGGCGCAGGTACTTCCACTGAAGCTAGTGAAGGTTCTGCATCAGGTAGCGAAACTGCTACTTCTGGTTCCGAAACTGCAGGTAGCGAAACCGCTACCTCTGGTTCCGAAACTGCAGGTACTTCTACCGAGGCTAGCGAAGGTTCTGCATCAGGTAGCACTGCTGGTTCCGAGACTTCTACTGAAGCAGGTACTAGCGAGTCCGCTACTAGCGAATCTGGCGCAGGTACTTCCACTGAAGCTAGTGAAGGTTCTGCATCAGGTAGCGAAACTGCTACTTCTGGTTCCGAAACTGCAGGTAGCACTGCTGGCTCCGAGACTTCTACCGAAGCAGGTAGCACTGCAGGTTCCGAAACTTCCACTGAAGCAGGTAGCGAAACTGCTACCTCTGGCTCTGAGACTGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCAGGTAGCGAAACCGCTACCTCTGGTTCCGAAACTGCAGGTACTTCTACCGAGGCTAGCGAAGGTTCTGCATCAGGTAGCACTGCTGGTTCCGAGACTTCTACTGAAGCAGGTAGCGAAACTGCTACTTCCGGCTCTGAGACTGCAGGTACTAGTGAATCCGCAACTAGCGAATCTGGCGCAGGTAGCACTGCAGGCTCTGAGACTTCCACTGAAGCAGGTAGCACTGCTGGTTCCGAAACCTCTACCGAAGCAGGTAGCACTGCAGGTTCTGAAACCTCCACTGAAGCAGGTACTTCCACTGAGGCTAGTGAAGGCTCTGCATCAGGTAGCACTGCTGGTTCCGAAACCTCTACCGAAGCAGGTAGCACTGCAGGTTCTGAAACCTCCACTGAAGCAGGTACTTCCACTGAGGCTAGTGAAGGCTCTGCATCAGGTAGCACTGCAGGTTCTGAGACTTCCACCGAAGCAGGTAGCGAAACTGCTACTTCTGGTTCCGAAACTGCAGGTACTTCCACTGAAGCTAGTGAAGGTTCCGCATCAGGTACTAGTGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACCGCAACCTCCGGTTCTGAAACTGCAGGTACTAGCGAATCCGCAACCAGCGAATCTGGCGCAGGTACTAGTGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACCGCAACCTCCGGTTCTGAAACTGCAGGTACTAGCGAATCCGCAACCAGCGAATCTGGCGCAGGTAGCGAAACTGCTACTTCCGGCTCTGAGACTGCAGGTACTTCCACCGAAGCAAGCGAAGGTTCCGCATCAGGTACTTCCACCGAGGCTAGTGAAGGCTCTGCATCAGGTAGCACTGCTGGCTCCGAGACTTCTACCGAAGCAGGTAGCACTGCAGGTTCCGAAACTTCCACTGAAGCAGGTAGCGAAACTGCTACCTCTGGCTCTGAGACTGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCAGGTAGCGAAACTGCTACTTCCGGCTCCGAGACTGCAGGTAGCGAAACTGCTACTTCTGGCTCCGAAACTGCAGGTACTTCTACTGAGGCTAGTGAAGGTTCCGCATCAGGTACTAGCGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACTGCTACCTCTGGCTCCGAGACTGCAGGTAGCGAAACTGCAACCTCTGGCTCTGAAACTGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCA

One may clone the library of XTEN-encoding genes into one or moreexpression vectors known in the art. To facilitate the identification ofwell-expressing library members, one can construct the library as fusionto a reporter protein. Non-limiting examples of suitable reporter genesare green fluorescent protein, luciferace, alkaline phosphatase, andbeta-galactosidase. By screening, one can identify short XTEN sequencesthat can be expressed in high concentration in the host organism ofchoice. Subsequently, one can generate a library of random XTEN dimersand repeat the screen for high level of expression. Subsequently, onecan screen the resulting constructs for a number of properties such aslevel of expression, protease stability, or binding to antiserum.

One aspect of the invention is to provide polynucleotide sequencesencoding the components of the fusion protein wherein the creation ofthe sequence has undergone codon optimization. Of particular interest iscodon optimization with the goal of improving expression of thepolypeptide compositions and to improve the genetic stability of theencoding gene in the production hosts. For example, codon optimizationis of particular importance for XTEN sequences that are rich in glycineor that have repetitive amino acid sequences. Codon optimization can beperformed using computer programs (Gustafsson, C., et al. (2004) TrendsBiotechnol, 22: 346-53), some of which minimize ribosomal pausing (CodaGenomics Inc.). In one embodiment, one can perform codon optimization byconstructing codon libraries where all members of the library encode thesame amino acid sequence but where codon usage is varied. Such librariescan be screened for highly expressing and genetically stable membersthat are particularly suitable for the large-scale production ofXTEN-containing products. When designing XTEN sequences one can considera number of properties. One can minimize the repetitiveness in theencoding DNA sequences. In addition, one can avoid or minimize the useof codons that are rarely used by the production host (e.g. the AGG andAGA arginine codons and one leucine codon in E. coli). In the case of E.coli, two glycine codons, GGA and GGG, are rarely used in highlyexpressed proteins. Thus codon optimization of the gene encoding XTENsequences can be very desirable. DNA sequences that have a high level ofglycine tend to have a high GC content that can lead to instability orlow expression levels. Thus, when possible, it is preferred to choosecodons such that the GC-content of XTEN-encoding sequence is suitablefor the production organism that will be used to manufacture the XTEN.

Optionally, the full-length XTEN-encoding gene may comprise one or moresequencing islands. In this context, sequencing islands areshort-stretch sequences that are distinct from the XTEN libraryconstruct sequences and that include a restriction site not present orexpected to be present in the full-length XTEN-encoding gene. In oneembodiment, a sequencing island is the sequence5′-AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGT-3′ (SEQ ID NO: 216). In anotherembodiment, a sequencing island is the sequence5′-AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGT-3′ (SEQ ID NO: 217).

As an alternative, one can construct codon libraries where all membersof the library encode the same amino acid sequence but where codon usageis varied. Such libraries can be screened for highly expressing andgenetically stable members that are particularly suitable for thelarge-scale production of XTEN-containing products.

Optionally, one can sequence clones in the library to eliminate isolatesthat contain undesirable sequences. The initial library of short XTENsequences can allow some variation in amino acid sequence. For instanceone can randomize some codons such that a number of hydrophilic aminoacids can occur in a particular position.

During the process of iterative multimerization one can screen theresulting library members for other characteristics like solubility orprotease resistance in addition to a screen for high-level expression.

In one embodiment of a construct encoding a binding fusion protein, oncethe gene that encodes the XTEN of desired length and properties isselected, it is genetically fused to the nucleotides encoding the N-and/or the C-terminus of the targeting moiety gene(s) by cloning it intothe construct adjacent and in frame with the gene coding for thetargeting moiety or adjacent to a linker sequence. The inventionprovides various permutations of the foregoing, depending on the bindingfusion protein to be encoded. For example, a gene encoding a bindingfusion protein comprising two targeting moieties such as embodied byformula II, as depicted above, would have polynucleotides encoding twotargeting moieties, a linker, at least a first XTEN, and optionally asecond XTEN. The step of cloning the targeting moiety genes into theXTEN construct can occur through a ligation or multimerization step. Asshown in FIG. 6A-FIG. 6D, the constructs encoding binding fusionproteins can be designed in different configurations of the components;e.g., XTEN 205, VL 202, VH 204 and linker sequences 203 or 206. In oneembodiment, as illustrated in FIG. 6A, the construct comprisespolynucleotide sequences complementary to, or those that encode amonomeric polypeptide of components in the following order (5′ to 3′) VL202, linker 203, VH 204, and XTEN 205, or the reverse order. As will beapparent to those of skill in the art, in view of the disclosure andFIG. 6A-FIG. 6D, other permutations or combinations of the foregoing arepossible.

The invention also encompasses polynucleotides comprising XTEN-encodingpolynucleotide variants that have a high percentage of sequence identityto (a) a polynucleotide sequence from Table 10, or (b) sequences thatare complementary to the polynucleotides of (a). A polynucleotide with ahigh percentage of sequence identity is one that has at least about an80% nucleic acid sequence identity, alternatively at least about 81%,alternatively at least about 82%, alternatively at least about 83%,alternatively at least about 84%, alternatively at least about 85%,alternatively at least about 86%, alternatively at least about 87%,alternatively at least about 88%, alternatively at least about 89%,alternatively at least about 90%, alternatively at least about 91%,alternatively at least about 92%, alternatively at least about 93%,alternatively at least about 94%, alternatively at least about 95%,alternatively at least about 96%, alternatively at least about 97%,alternatively at least about 98%, and alternatively at least about 99%nucleic acid sequence identity to (a) or (b) of the foregoing, or thatcan hybridize with the target polynucleotide or its complement understringent conditions.

Homology, sequence similarity or sequence identity of nucleotide oramino acid sequences may also be determined conventionally by usingknown software or computer programs such as the BestFit or Gap pairwisecomparison programs (GCG Wisconsin Package, Genetics Computer Group, 575Science Drive, Madison, Wis. 53711). BestFit uses the local homologyalgorithm of Smith and Waterman (Advances in Applied Mathematics. 1981.2: 482-489), to find the best segment of identity or similarity betweentwo sequences. Gap performs global alignments: all of one sequence withall of another similar sequence using the method of Needleman andWunsch, (Journal of Molecular Biology. 1970. 48:443-453). When using asequence alignment program such as BestFit, to determine the degree ofsequence homology, similarity or identity, the default setting may beused, or an appropriate scoring matrix may be selected to optimizeidentity, similarity or homology scores.

Nucleic acid sequences that are “complementary” are those that arecapable of base-pairing according to the standard Watson-Crickcomplementarity rules. As used herein, the term “complementarysequences” means nucleic acid sequences that are substantiallycomplementary, as may be assessed by the same nucleotide comparison setforth above, or as defined as being capable of hybridizing to thepolynucleotides that encode the binding fusion protein sequences understringent conditions, such as those described herein.

The resulting polynucleotides encoding the binding fusion proteinchimeric compositions can then be individually cloned into an expressionvector. The nucleic acid sequence may be inserted into the vector by avariety of procedures. In general, DNA is inserted into an appropriaterestriction endonuclease site(s) using techniques known in the art.Vector components generally include, but are not limited to, one or moreof a signal sequence, an origin of replication, one or more markergenes, an enhancer element, a promoter, and a transcription terminationsequence. Construction of suitable vectors containing one or more ofthese components employs standard ligation techniques that are known tothe skilled artisan. Such techniques are well known in the art and welldescribed in the scientific and patent literature.

Various vectors are publicly available. The vector may, for example, bein the form of a plasmid, cosmid, viral particle, or phage. Bothexpression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchvector sequences are well known for a variety of bacteria, yeast, andviruses. Useful expression vectors that can be used include, forexample, segments of chromosomal, non-chromosomal and synthetic DNAsequences. Suitable vectors include, but are not limited to, derivativesof SV40 and pcDNA and known bacterial plasmids such as col EI, pCR1,pBR322, pMal-C2, pET, pGEX as described by Smith, et al., Gene 57:31-40(1988), pMB9 and derivatives thereof, plasmids such as RP4, phage DNAssuch as the numerous derivatives of phage I such as NM98 9, as well asother phage DNA such as M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2 micron plasmid or derivatives of the 2 mplasmid, as well as centomeric and integrative yeast shuttle vectors;vectors useful in eukaryotic cells such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orthe expression control sequences; and the like. The requirements arethat the vectors are replicable and viable in the host cell of choice.Low- or high-copy number vectors may be used as desired.

Promoters suitable for use in expression vectors with prokaryotic hostsinclude the β-lactamase and lactose promoter systems [Chang et al.,Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters suchas the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)]. Promoters for use in bacterial systems can also contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingbinding fusion protein.

For example, in a baculovirus expression system, both non-fusiontransfer vectors, such as, but not limited to pVL941 (BamHI cloningsite, available from Summers, et al., Virology 84:390-402 (1978)),pVL1393 (BamHI, Smal, Xbal, EcoRI, IVotl, Xmalll, BgIII and Pstl cloningsites; Invitrogen), pVL1392 (BgIII, Pstl, NotI, XmaIII, EcoRI, Xball,Smal and BamHI cloning site; Summers, et al., Virology 84:390-402 (1978)and Invitrogen) and pBlueBacIII (BamHI, BgIII, Pstl, Ncol and Hindi IIcloning site, with blue/white recombinant screening, Invitrogen), andfusion transfer vectors such as, but not limited to, pAc700 (BamHI andKpnl cloning sites, in which the BamHI recognition site begins with theinitiation codon; Summers, et al., Virology 84:390-402 (1978)), pAc701and pAc70-2 (same as pAc700, with different reading frames), pAc360[BamHI cloning site 36 base pairs downstream of a polyhedrin initiationcodon; Invitrogen (1995)) and pBlueBacHisA, B, C (three differentreading frames with BamH I, BgI II, Pstl, Nco l and Hind III cloningsite, an N-terminal peptide for ProBond purification and blue/whiterecombinant screening of plaques; Invitrogen (220) can be used.

Schematics of exemplary plasmids containing one or more of thecomponents described above are illustrated in FIG. 10A-FIG. 10D.

Mammalian expression vectors can comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements Mammalian expression vectors contemplated for use inthe invention include vectors with inducible promoters, such as thedihydrofolate reductase promoters, any expression vector with a DHFRexpression cassette or a DHFR/methotrexate co-amplification vector suchas pED (Pstl, Sail, Sbal, Smal and EcoRI cloning sites, with the vectorexpressing both the cloned gene and DHFR; Randal J. Kaufman, 1991,Randal J. Kaufman, Current Protocols in Molecular Biology, 16,12(1991)). Alternatively a glutamine synthetase/methionine sulfoximineco-amplification vector, such as pEE14 (Hindlll, Xball, Smal, Sbal,EcoRI and Sell cloning sites in which the vector expresses glutaminesynthetase and the cloned gene; Celltech). A vector that directsepisomal expression under the control of the Epstein Barr Virus (EBV) ornuclear antigen (EBNA) can be used such as pREP4 (BamHI r SfH, Xhol,NotI, Nhel, Hindi II, Nhel, PvuII and Kpnl cloning sites, constitutiveRSV-LTR promoter, hygromycin selectable marker; Invitrogen), pCEP4(BamHI, SfH, Xhol, NotI, Nhel, Hindlll, Nhel, PvuII and Kpnl cloningsites, constitutive hCMV immediate early gene promoter, hygromycinselectable marker; Invitrogen), pMEP4 (.Kpnl, Pvul, Nhel, Hindlll, NotI,Xhol, Sfil, BamHI cloning sites, inducible methallothionein H a genepromoter, hygromycin selectable marker, Invitrogen), pREP8 (BamHI, Xhol,NotI, Hindlll, Nhel and Kpnl cloning sites, RSV-LTR promoter, histidinolselectable marker; Invitrogen), pREP9 (Kpnl, Nhel, Hind 111, NotI, Xhol, Sfi l, BamH I cloning sites, RSV-LTR promoter, G418 selectablemarker; Invitrogen), and pEBVHis (RSV-LTR promoter, hygromycinselectable marker, N-terminal peptide purifiable via ProBond resin andcleaved by enterokinase; Invitrogen).

Selectable mammalian expression vectors for use in the inventioninclude, but are not limited to, pRc/CMV (Hind lll, BstXI, NotI, Sbaland Apal cloning sites, G418 selection, Invitrogen), pRc/RSV (Hind II,Spel, BstXI, NotI, Xbal cloning sites, G418 selection, Invitrogen) andthe like. Vaccinia virus mammalian expression vectors (see, for example,Randall J. Kaufman, Current Protocols in Molecular Biology 16.12(Frederick M. Ausubel, et al., eds. Wiley 1991) that can be used in thepresent invention include, but are not limited to, pSC11 (Smal cloningsite, TK- and beta-gal selection), pMJ601 (Sal 1, Sma 1, A fII, Narl,BspMlI, BamHI, Apal, Nhel, SacII, Kpnl and Hindlll cloning sites; TK-and -gal selection), pTKgptF1S (EcoRI, Pstl, SaIII, Accl, HindII, Sbal,BamHI and Hpa cloning sites, TK or XPRT selection) and the like.

Yeast expression systems that can also be used in the present inventioninclude, but are not limited to, the non-fusion pYES2 vector (XJbal,Sphl, Shol, NotI, GstXI, EcoRI, BstXI, BamHI, Sad, Kpnl and Hindlllcloning sites, Invitrogen), the fusion pYESHisA, B, C (Xball, Sphl,Shol, Nod, BstXI, EcoRI, BamHI, Sad, Kpnl and Hindi II cloning sites,N-terminal peptide purified with ProBond resin and cleaved withenterokinase; Invitrogen), pRS vectors and the like.

In addition, the expression vector containing the chimeric bindingfusion protein-encoding polynucleotide molecule may include drugselection markers. Such markers aid in cloning and in the selection oridentification of vectors containing chimeric DNA molecules. Forexample, genes that confer resistance to neomycin, puromycin,hygromycin, dihydrofolate reductase (DHFR) inhibitor, guaninephosphoribosyl transferase (GPT), zeocin, and histidinol are usefulselectable markers. Alternatively, enzymes such as herpes simplex virusthymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may beemployed. Immunologic markers also can be employed. Any known selectablemarker may be employed so long as it is capable of being expressedsimultaneously with the nucleic acid encoding a gene product. Furtherexamples of selectable markers are well known to one of skill in the artand include reporters such as enhanced green fluorescent protein (EGFP),beta-galactosidase (β-gal) or chloramphenicol acetyltransferase (CAT).

In one embodiment, the polynucleotide encoding a binding fusion proteincomposition can be fused C-terminally to an N-terminal signal sequenceappropriate for the expression host system. Signal sequences aretypically proteolytically removed from the protein during thetranslocation and secretion process, generating a defined N-terminus. Awide variety of signal sequences have been described for most expressionsystems, including bacterial, yeast, insect, and mammalian systems. Anon-limiting list of preferred examples for each expression systemfollows herein. Preferred signal sequences are OmpA, PhoA, and DsbA forE. coli expression. Signal peptides preferred for yeast expression areppL-alpha, DEX4, invertase signal peptide, acid phosphatase signalpeptide, CPY, or INU1. For insect cell expression the preferred signalsequences are sexta adipokinetic hormone precursor, CP1, CP2, CP3, CP4,TPA, PAP, or gp67. For mammalian expression the preferred signalsequences are IL2L, SV40, IgG kappa and IgG lambda.

In another embodiment, a leader sequence, potentially comprising awell-expressed, independent protein domain, can be fused to theN-terminus of the binding fusion protein sequence, separated by aprotease cleavage site. While any leader peptide sequence that does notinhibit cleavage at the designed proteolytic site can be used, sequencesin preferred embodiments will comprise stable, well-expressed sequencessuch that expression and folding of the overall composition is notsignificantly adversely affected, and preferably expression, solubility,and/or folding efficiency are significantly improved. A wide variety ofsuitable leader sequences have been described in the literature. Anon-limiting list of suitable sequences includes maltose bindingprotein, cellulose binding domain, glutathione S-transferase, 6×His tag(SEQ ID NO: 218), FLAG tag, hemaglutinin tag, and green fluorescentprotein. The leader sequence can also be further improved by codonoptimization, especially in the second codon position following the ATGstart codon, by methods well described in the literature andhereinabove.

Various in vitro enzymatic methods for cleaving proteins at specificsites are known. Such methods include use of enterokinase (DDDK) (SEQ IDNO: 219), Factor Xa (IDGR) (SEQ ID NO: 880), thrombin (LVPRGS) (SEQ IDNO: 220), PreScission™ (LEVLFQGP) (SEQ ID NO: 221), TEV protease(EQLYFQG) (SEQ ID NO: 222), 3C protease (ETLFQGP) (SEQ ID NO: 223),Sortase A (LPETG) (SEQ ID NO: 224), Granzyme B (D/X, N/X, M/N or SA),inteins, SUMO, DAPase (TAGZyme™), Aeromonas aminopeptidase,Aminopeptidase M, and carboxypeptidases A and B. Additional methods aredisclosed in Arnau, et al., Protein Expression and Purification 48: 1-13(2006).

In other embodiments, an optimized polynucleotide sequence encoding atleast about 20 to about 60 amino acids with XTEN characteristics can beincluded at the N-terminus of the XTEN sequence to promote theinitiation of translation to allow for expression of XTEN fusions at theN-terminus of proteins without the presence of a helper domain. In anadvantage of the foregoing, the sequence does not require subsequentcleavage, thereby reducing the number of steps to manufactureXTEN-containing compositions. As described in more detail in theExamples, the optimized N-terminal sequence has attributes of anunstructured protein, but may include nucleotide bases encoding aminoacids selected for their ability to promote initiation of translationand enhanced expression. In one embodiment of the foregoing, theoptimized polynucleotide encodes an XTEN sequence with at least about90% sequence identity to AE624. In another embodiment of the foregoing,the optimized polynucleotide encodes an XTEN sequence with at leastabout 90% sequence identity to AE912. In yet another embodiment of theforegoing, the optimized polynucleotide encodes an XTEN sequence with atleast about 90% sequence identity to AM923.

In another embodiment, the protease site of the leader sequenceconstruct is chosen such that it is recognized by an in vivo protease.In this embodiment, the protein is purified from the expression systemwhile retaining the leader by avoiding contact with an appropriateprotease. The full-length construct is then injected into a patient.Upon injection, the construct comes into contact with the proteasespecific for the cleavage site and is cleaved by the protease. In thecase where the uncleaved protein is substantially less active than thecleaved form, this method has the beneficial effect of allowing higherinitial doses while avoiding toxicity, as the active form is generatedslowly in vivo. Some non-limiting examples of in vivo proteases whichare useful for this application include tissue FXIa, FXIIa, kallikrein,FVIIa, FIXa, FXa, FIIa (thrombin), Elastase-2, granzyme B, MMP-12,MMP-13, MMP-17 or MMP-20, or by non-mammalian proteases such as TEV,enterokinase, Pre Scission™ protease (rhinovirus 3C protease), andsortase A.

In this manner, a chimeric DNA molecule coding for a monomeric bindingfusion protein is generated within the construct. Optionally, thischimeric DNA molecule may be transferred or cloned into anotherconstruct that is a more appropriate expression vector. At this point, ahost cell capable of expressing the chimeric DNA molecule can betransformed with the chimeric DNA molecule. The vectors containing theDNA segments of interest can be transferred into the host cell bywell-known methods, depending on the type of cellular host. For example,calcium chloride transfection is commonly utilized for prokaryoticcells, whereas calcium phosphate treatment, lipofection, orelectroporation may be used for other cellular hosts. Other methods usedto transform mammalian cells include the use of polybrene, protoplastfusion, liposomes, electroporation, and microinjection. See, generally,Sambrook, et al., supra.

The transformation may occur with or without the utilization of acarrier, such as an expression vector. Then, the transformed host cellis cultured under conditions suitable for expression of the chimeric DNAmolecule encoding the binding fusion protein.

The present invention also provides a host cell for expressing themonomeric fusion protein compositions disclosed herein. Examples ofsuitable eukaryotic host cells include, but are not limited to mammaliancells, such as VERO cells, HELA cells such as ATCC No. CCL2, CHO celllines, COS cells, WI38 cells, BHK cells, HepG2 cells, 3T3 cells, A549cells, PC12 cells, K562 cells, 293 cells, Sf9 cells and CvI cells.Examples of suitable non-mammalian eukaryotic cells include eukaryoticmicrobes such as filamentous fungi or yeast are suitable cloning orexpression hosts for encoding vectors. Saccharomyces cerevisiae is acommonly used lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 737 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K.drosophilarum (ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135(1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226);Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol.,28:265-278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene,26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Other suitable cells that can be used in the present invention include,but are not limited to, prokaryotic host cells strains such asEscherichia coli, (e.g., strain DH5-α), Bacillus subtilis, Salmonellatyphimurium, or strains of the genera of Pseudomonas, Streptomyces andStaphylococcus. Non-limiting examples of suitable prokaryotes includethose from the genera: Actinoplanes; Archaeoglobus; Bdellovibrio;Borrelia; Chloroflexus; Enterococcus; Escherichia; Lactobacillus;Listeria; Oceanobacillus; Paracoccus; Pseudomonas; Staphylococcus;Streptococcus; Streptomyces; Thermoplasma; and Vibrio. Non-limitingexamples of specific strains include: Archaeoglobus fulgidus;Bdellovibrio bacteriovorus; Borrelia burgdorferi; Chloroflexusaurantiacus; Enterococcus faecalis; Enterococcus faecium; Lactobacillusjohnsonii; Lactobacillus plantarum; Lactococcus lactis; Listeriainnocua; Listeria monocytogenes; Oceanobacillus iheyensis; Paracoccuszeaxanthinifaciens; Pseudomonas mevalonii; Staphylococcus aureus;Staphylococcus epidermidis; Staphylococcus haemolyticus; Streptococcusagalactiae; Streptomyces griseolosporeus; Streptococcus mutans;Streptococcus pneumoniae; Streptococcus pyogenes; Thermoplasmaacidophilum; Thermoplasma volcanium; Vibrio cholerae; Vibrioparahaemolyticus; and Vibrio vulnificus.

Host cells containing the polynucleotides of interest can be cultured inconventional nutrient media (e.g., Ham's nutrient mixture) modified asappropriate for activating promoters, selecting transformants oramplifying genes. The culture conditions, such as temperature, pH andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification. For compositions secreted by the host cells, supernatantfrom centrifugation is separated and retained for further purification.Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, all of which arewell known to those skilled in the art. Embodiments that involve celllysis may entail use of a buffer that contains protease inhibitors thatlimit degradation after expression of the chimeric DNA molecule.Suitable protease inhibitors include, but are not limited to leupeptin,pepstatin or aprotinin. The supernatant then may be precipitated insuccessively increasing concentrations of saturated ammonium sulfate.

Gene expression may be measured in a sample directly, for example, byconventional Southern blotting, Northern blotting to quantitate thetranscription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205(1980)], dot blotting (DNA analysis), or in situ hybridization, using anappropriately labeled probe, based on the sequences provided herein.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to a surface,so that upon the formation of duplex on the surface, the presence ofantibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunological offluorescent methods, such as immunohistochemical staining of cells ortissue sections and assay of cell culture or body fluids or thedetection of selectable markers, to quantitate directly the expressionof gene product. Antibodies useful for immunohistochemical stainingand/or assay of sample fluids may be either monoclonal or polyclonal,and may be prepared in any mammal Conveniently, the antibodies may beprepared against a native sequence polypeptide or against a syntheticpeptide based on the DNA sequences provided herein or against exogenoussequence fused to targeting moieties and encoding a specific epitope.Examples of selectable markers are well known to one of skill in the artand include reporters such as enhanced green fluorescent protein (EGFP),beta-galactosidase (β-gal) or chloramphenicol acetyltransferase (CAT).

Expressed binding fusion protein product(s) may be purified via methodsknown in the art or by methods disclosed herein. Procedures such as gelfiltration, affinity purification, salt fractionation, ion exchangechromatography, size exclusion chromatography, hydroxyapatite adsorptionchromatography, hydrophobic interaction chromatography and gelelectrophoresis may be used; each tailored to recover and purify thefusion protein produced by the respective host cells. Some expressedbinding fusion protein may require refolding during isolation andpurification. Methods of purification are described in Robert K. Scopes,Protein Purification: Principles and Practice, Charles R. Castor (ed.),Springer-Verlag 1994, and Sambrook, et al., supra. Multi-steppurification separations are also described in Baron, et al., Crit. Rev.Biotechnol. 10:179-90 (1990) and Below, et al., J. Chromatogr. A.679:67-83 (1994).

IV). Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprisingbinding fusion proteins, XTEN-drug conjugates, or BFP-D conjugates. Inone embodiment, the pharmaceutical composition comprises the bindingfusion protein and at least one pharmaceutically acceptable carrier. Inanother embodiment, the pharmaceutical composition comprises the BFP-Dand at least one pharmaceutically acceptable carrier. In anotherembodiment, the pharmaceutical composition comprises the XTEN-drugconjugate and at least one pharmaceutically acceptable carrier. Thepharmaceutical compositions of the present invention can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby the polypeptide is combined in admixture with apharmaceutically acceptable carrier vehicle, such as aqueous solutionsor buffers, pharmaceutically acceptable suspensions and emulsions.Examples of non-aqueous solvents include propyl ethylene glycol,polyethylene glycol and vegetable oils. Therapeutic formulations areprepared for storage by mixing the active ingredient having the desireddegree of purity with optional physiologically acceptable carriers,excipients or stabilizers, as described in Remington's PharmaceuticalSciences 16th edition, Osol, A. Ed. (1980), in the form of lyophilizedformulations or aqueous solutions. In addition, the pharmaceuticalcompositions can also contain other pharmaceutically active compounds ora plurality of compositions of the invention.

The pharmaceutical compositions may be administered for therapy by anysuitable route including oral, rectal, nasal, topical (includingtransdermal, aerosol, buccal and sublingual), vaginal, parenteral(including subcutaneous, subcutaneous by infusion pump, intramuscular,intravenous and intradermal), intravitreal, and pulmonary. It will alsobe appreciated that the preferred route will vary with the condition andage of the recipient, and the disease being treated.

In preferred embodiments, the pharmaceutical composition is administeredparenterally. In this embodiment, the composition may be supplied as alyophilized powder to be reconstituted prior to administration. Thecomposition may also be supplied in a liquid form, which can beadministered directly to a patient. In one embodiment, the compositionis supplied as a liquid in a pre-filled syringe such that a patient caneasily self-administer the composition.

The compositions of the invention may be formulated using a variety ofexcipients. Suitable excipients include microcrystalline cellulose (e.g.Avicel PH102, Avicel PH101), polymethacrylate, poly(ethyl acrylate,methyl methacrylate, trimethylammonioethyl methacrylate chloride) (suchas Eudragit RS-30D), hydroxypropyl methylcellulose (Methocel K100M,Premium CR Methocel K100M, Methocel E5, Opadry®), magnesium stearate,talc, triethyl citrate, aqueous ethylcellulose dispersion (Surelease®),and protamine sulfate. The slow release agent may also comprise acarrier, which can comprise, for example, solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents. Pharmaceutically acceptable salts can also be used inthese slow release agents, for example, mineral salts such ashydrochlorides, hydrobromides, phosphates, or sulfates, as well as thesalts of organic acids such as acetates, proprionates, malonates, orbenzoates. The composition may also contain liquids, such as water,saline, glycerol, and ethanol, as well as substances such as wettingagents, emulsifying agents, or pH buffering agents. Liposomes may alsobe used as a carrier.

For liquid formulations, a desired property is that the formulation besupplied in a form that can pass through a 25, 28, 30, 31, 32 gaugeneedle for intravenous, intramuscular, intraarticular, or subcutaneousadministration. Syringe pumps may also be used to delivery thepharmaceutical compositions of the invention. Such devices are describedin U.S. Pat. Nos. 4,976,696; 4,933,185; 5,017,378; 6,309,370; 6,254,573;4,435,173; 4,398,908; 6,572,585; 5,298,022; 5,176,502; 5,492,534;5,318,540; and 4,988,337, the contents of which are incorporated hereinby reference. One skilled in the art, considering both the disclosure ofthis invention and the disclosures of these other patents could producea syringe pump for the extended release of the compositions of thepresent invention.

Administration via transdermal formulations can be performed usingmethods also known in the art, including those described generally in,e.g., U.S. Pat. Nos. 5,186,938 and 6,183,770, 4,861,800, 6,743,211,6,945,952, 4,284,444, and WO 89/09051, incorporated herein by referencein their entireties. A transdermal patch is a particularly usefulembodiment with polypeptides having absorption problems. Patches can bemade to control the release of skin-permeable active ingredients over a12 hour, 24 hour, 3 day, and 7 day period. In one example, a 2-folddaily excess of a polypeptide of the present invention is placed in anon-volatile fluid. The compositions of the invention are provided inthe form of a viscous, non-volatile liquid. The penetration through skinof specific formulations may be measures by standard methods in the art(for example, Franz et al., J. Invest. Derm. 64:194-195 (1975)).Examples of suitable patches are passive transfer skin patches,iontophoretic skin patches, or patches with microneedles such asNicoderm.

In other embodiments, the composition may be delivered via intranasal,buccal, or sublingual routes to the brain to enable transfer of theactive agents through the olfactory passages into the CNS and reducingthe systemic administration. Devices commonly used for this route ofadministration are included in U.S. Pat. No. 6,715,485. Compositionsdelivered via this route may enable increased CNS dosing or reducedtotal body burden reducing systemic toxicity risks associated withcertain drugs. Preparation of a pharmaceutical composition for deliveryin a subdermally implantable device can be performed using methods knownin the art, such as those described in, e.g., U.S. Pat. Nos. 3,992,518;5,660,848; and 5,756,115.

V). Pharmaceutical Kits

In another aspect, the invention provides a kit to facilitate the use ofthe composition embodiments disclosed herein. In one embodiment, the kitcomprises, in at least a first container: (a) an amount of a bindingfusion protein composition sufficient to administer in treatment of asubject with a disease, condition or disorder; and (b) an amount of apharmaceutically acceptable carrier; together in a formulation ready forinjection or for reconstitution with sterile water, buffer, or dextrose;together with a label identifying the binding fusion protein drug andstorage and handling conditions, and/or a sheet of the approvedindications for the drug and instructions for the reconstitution and/oradministration of the binding fusion protein drug for the use for theprevention and/or treatment of a approved indication, appropriate dosageand safety information, and information identifying the lot andexpiration of the drug.

In another embodiment, the kit comprises, in at least a first container:(a) an amount of a binding fusion protein-drug conjugate compositionsufficient to administer in treatment of a subject with a disease,condition or disorder; and (b) an amount of a pharmaceuticallyacceptable carrier; together in a formulation ready for injection or forreconstitution with sterile water, buffer, or dextrose; together with alabel identifying the binding fusion protein-drug conjugate and storageand handling conditions, and/or a sheet of the approved indications forthe drug and instructions for the reconstitution and/or administrationof the compositions for the use for the prevention and/or treatment of aapproved indication, appropriate dosage and safety information, andinformation identifying the lot and expiration of the drug.

In another embodiment, the kit comprises, in at least a first container:(a) an amount of an XTEN-drug conjugate composition sufficient toadminister in treatment of a subject with a disease, condition ordisorder; and (b) an amount of a pharmaceutically acceptable carrier;together in a formulation ready for injection or for reconstitution withsterile water, buffer, or dextrose; together with a label identifyingthe XTEN-drug conjugate and storage and handling conditions, and/or asheet of the approved indications for the drug and instructions for thereconstitution and/or administration of the compositions for the use forthe prevention and/or treatment of a approved indication, appropriatedosage and safety information, and information identifying the lot andexpiration of the drug.

In any of the embodiments of the foregoing kits, the kit can comprise asecond container that can carry a suitable diluent for the subjectcomposition, which will provide the user with the appropriateconcentration of the pharmaceutical composition to be delivered to thesubject.

EXAMPLES Example 1: Construction of XTEN_AD36 Motif Segments

The following example describes the construction of a collection ofcodon-optimized genes encoding motif sequences of 36 amino acids. As afirst step, a stuffer vector pCW0359 was constructed based on a pETvector and that includes a T7 promoter. pCW0359 encodes a cellulosebinding domain (CBD) and a TEV protease recognition site followed by astuffer sequence that is flanked by BsaI, BbsI, and KpnI sites. The BsaIand BbsI sites were inserted such that they generate compatibleoverhangs after digestion. The stuffer sequence is followed by atruncated version of the GFP gene and a His tag. The stuffer sequencecontains stop codons and thus E. coli cells carrying the stuffer plasmidpCW0359 form non-fluorescent colonies. The stuffer vector pCW0359 wasdigested with BsaI and KpnI to remove the stuffer segment and theresulting vector fragment was isolated by agarose gel purification. Thesequences were designated XTEN_AD36, reflecting the AD family of motifs.Its segments have the amino acid sequence [X]₃ where X is a 12merpeptide with the sequences: GESPGGSSGSES (SEQ ID NO: 2), GSEGSSGPGESS(SEQ ID NO: 3), GSSESGSSEGGP (SEQ ID NO: 4), or GSGGEPSESGSS (SEQ ID NO:5). The insert was obtained by annealing the following pairs ofphosphorylated synthetic oligonucleotide pairs:

AD1for: (SEQ ID NO: 225) AGGTGAATCTCCDGGTGGYTCYAGCGGTTCYGARTC AD1rev:(SEQ ID NO: 226) ACCTGAYTCRGAACCGCTRGARCCACCHGGAGATTC AD2for:(SEQ ID NO: 227) AGGTAGCGAAGGTTCTTCYGGTCCDGGYGARTCYTC AD2rev:(SEQ ID NO: 228) ACCTGARGAYTCRCCHGGACCRGAAGAACCTTCGCT AD3for:(SEQ ID NO: 229) AGGTTCYTCYGAAAGCGGTTCTTCYGARGGYGGTCC AD3rev:(SEQ ID NO: 230) ACCTGGACCRCCYTCRGAAGAACCGCTTTCRGARGA AD4for:(SEQ ID NO: 231) AGGTTCYGGTGGYGAACCDTCYGARTCTGGTAGCTC

We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor:AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 232) and the non-phosphorylatedoligonucleotide pr_3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 233).The annealed oligonucleotide pairs were ligated, which resulted in amixture of products with varying length that represents the varyingnumber of 12mer repeats ligated to one BbsI/KpnI segment. The productscorresponding to the length of 36 amino acids were isolated from themixture by preparative agarose gel electrophoresis and ligated into theBsaI/KpnI digested stuffer vector pCW0359. Most of the clones in theresulting library designated LCW0401 showed green fluorescence afterinduction, which shows that the sequence of XTEN_AD36 had been ligatedin frame with the GFP gene and that most sequences of XTEN_AD36 had goodexpression levels.

We screened 96 isolates from library LCW0401 for high level offluorescence by stamping them onto agar plate containing IPTG. The sameisolates were evaluated by PCR and 48 isolates were identified thatcontained segments with 36 amino acids as well as strong fluorescence.These isolates were sequenced and 39 clones were identified thatcontained correct XTEN_AD36 segments. The file names of the nucleotideand amino acid constructs and the sequences for these segments arelisted in Table 11.

TABLE 11 DNA and Amino Acid Sequences for 36-mer motifs SEQ ID SEQ IDFile name Amino acid sequence NO: Nucleotide sequence NO: LCW0401_001_GSGGEPSESGSSGESP 234 GGTTCTGGTGGCGAACCGTCCGAGTCTGGTAG 272 GFP-N_A01.ab1GGSSGSESGESPGGSS CTCAGGTGAATCTCCGGGTGGCTCTAGCGGTT GSESCCGAGTCAGGTGAATCTCCTGGTGGTTCCAGC GGTTCCGAGTCA LCW0401_002_GSEGSSGPGESSGESP 235 GGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTC 273 GFP-N_B01.ab1GGSSGSESGSSESGSS TTCAGGTGAATCTCCTGGTGGTTCCAGCGGTTC EGGPTGAATCAGGTTCCTCCGAAAGCGGTTCTTCCG AGGGCGGTCCA LCW0401_003_GSSESGSSEGGPGSSE 236 GGTTCCTCTGAAAGCGGTTCTTCCGAAGGTGG 274 GFP-N_C01.ab1SGSSEGGPGESPGGSS TCCAGGTTCCTCTGAAAGCGGTTCTTCTGAGGG GSESTGGTCCAGGTGAATCTCCGGGTGGCTCCAGCG GTTCCGAGTCA LCW0401_004_GSGGEPSESGSSGSSE 237 GGTTCCGGTGGCGAACCGTCTGAATCTGGTAG 275 GFP-N_D01.ab1SGSSEGGPGSGGEPSE CTCAGGTTCTTCTGAAAGCGGTTCTTCCGAGGG SGSSTGGTCCAGGTTCTGGTGGTGAACCTTCCGAGTC TGGTAGCTCA LCW0401_007_GSSESGSSEGGPGSEG 238 GGTTCTTCCGAAAGCGGTTCTTCTGAGGGTGGT 276 GFP-N_F01.ab1SSGPGESSGSEGSSGP CCAGGTAGCGAAGGTTCTTCCGGTCCAGGTGA GESSGTCTTCAGGTAGCGAAGGTTCTTCTGGTCCTGG TGAATCTTCA LCW0401_008_GSSESGSSEGGPGESP 239 GGTTCCTCTGAAAGCGGTTCTTCCGAGGGTGG 277 GFP-N_G01.ab1GGSSGSESGSEGSSGP TCCAGGTGAATCTCCAGGTGGTTCCAGCGGTT GESSCTGAGTCAGGTAGCGAAGGTTCTTCTGGTCCA GGTGAATCCTCA LCW0401_012_GSGGEPSESGSSGSGG 240 GGTTCTGGTGGTGAACCGTCTGAGTCTGGTAG 278 GFP-N_H01.ab1EPSESGSSGSEGSSGP CTCAGGTTCCGGTGGCGAACCATCCGAATCTG GESSGTAGCTCAGGTAGCGAAGGTTCTTCCGGTCCA GGTGAGTCTTCA LCW0401_015_GSSESGSSEGGPGSEG 241 GGTTCTTCCGAAAGCGGTTCTTCCGAAGGCGG 279 GFP-N_A02.ab1SSGPGESSGESPGGSS TCCAGGTAGCGAAGGTTCTTCTGGTCCAGGCG GSESAATCTTCAGGTGAATCTCCTGGTGGCTCCAGC GGTTCTGAGTCA LCW0401_016_GSSESGSSEGGPGSSE 242 GGTTCCTCCGAAAGCGGTTCTTCTGAGGGCGG 280 GFP-N_B02.ab1SGSSEGGPGSSESGSS TCCAGGTTCCTCCGAAAGCGGTTCTTCCGAGG EGGPGCGGTCCAGGTTCTTCTGAAAGCGGTTCTTCCG AGGGCGGTCCA LCW0401_020_GSGGEPSESGSSGSEG 243 GGTTCCGGTGGCGAACCGTCCGAATCTGGTAG 281 GFP-N_E02.ab1SSGPGESSGSSESGSS CTCAGGTAGCGAAGGTTCTTCTGGTCCAGGCG EGGPAATCTTCAGGTTCCTCTGAAAGCGGTTCTTCTG AGGGCGGTCCA LCW0401_022_GSGGEPSESGSSGSSE 244 GGTTCTGGTGGTGAACCGTCCGAATCTGGTAG 282 GFP-N_F02.ab1SGSSEGGPGSGGEPSE CTCAGGTTCTTCCGAAAGCGGTTCTTCTGAAGG SGSSTGGTCCAGGTTCCGGTGGCGAACCTTCTGAAT CTGGTAGCTCA LCW0401_024_GSGGEPSESGSSGSSE 245 GGTTCTGGTGGCGAACCGTCCGAATCTGGTAG 283 GFP-N_G02.ab1SGSSEGGPGESPGGSS CTCAGGTTCCTCCGAAAGCGGTTCTTCTGAAG GSESGTGGTCCAGGTGAATCTCCAGGTGGTTCTAGC GGTTCTGAATCA LCW0401_026_GSGGEPSESGSSGESP 246 GGTTCTGGTGGCGAACCGTCTGAGTCTGGTAG 284 GFP-N_H02.ab1GGSSGSESGSEGSSGP CTCAGGTGAATCTCCTGGTGGCTCCAGCGGTTC GESSTGAATCAGGTAGCGAAGGTTCTTCTGGTCCTG GTGAATCTTCA LCW0401_027_GSGGEPSESGSSGESP 247 GGTTCCGGTGGCGAACCTTCCGAATCTGGTAG 285 GFP-N_A03.ab1GGSSGSESGSGGEPSE CTCAGGTGAATCTCCGGGTGGTTCTAGCGGTTC SGSSTGAGTCAGGTTCTGGTGGTGAACCTTCCGAGT CTGGTAGCTCA LCW0401_028_GSSESGSSEGGPGSSE 248 GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGG 286 GFP-N_B03.ab1SGSSEGGPGSSESGSS TCCAGGTTCTTCCGAAAGCGGTTCTTCCGAGG EGGPGCGGTCCAGGTTCTTCCGAAAGCGGTTCTTCTG AAGGCGGTCCA LCW0401_030_GESPGGSSGSESGSEG 249 GGTGAATCTCCGGGTGGCTCCAGCGGTTCTGA 287 GFP-N_C03.ab1SSGPGESSGSEGSSGP GTCAGGTAGCGAAGGTTCTTCCGGTCCGGGTG GESSAGTCCTCAGGTAGCGAAGGTTCTTCCGGTCCT GGTGAGTCTTCA LCW0401_031_GSGGEPSESGSSGSGG 250 GGTTCTGGTGGCGAACCTTCCGAATCTGGTAG 288 GFP-N_D03.ab1EPSESGSSGSSESGSS CTCAGGTTCCGGTGGTGAACCTTCTGAATCTGG EGGPTAGCTCAGGTTCTTCTGAAAGCGGTTCTTCCGA GGGCGGTCCA LCW0401_033_GSGGEPSESGSSGSGG 251 GGTTCCGGTGGTGAACCTTCTGAATCTGGTAG 289 GFP-N_E03.ab1EPSESGSSGSGGEPSE CTCAGGTTCCGGTGGCGAACCATCCGAGTCTG SGSSGTAGCTCAGGTTCCGGTGGTGAACCATCCGAG TCTGGTAGCTCA LCW0401_037_GSGGEPSESGSSGSSE 252 GGTTCCGGTGGCGAACCTTCTGAATCTGGTAG 290 GFP-N_F03.ab1SGSSEGGPGSEGSSGP CTCAGGTTCCTCCGAAAGCGGTTCTTCTGAGG GESSGCGGTCCAGGTAGCGAAGGTTCTTCTGGTCCG GGCGAGTCTTCA LCW0401_038_GSGGEPSESGSSGSEG 253 GGTTCCGGTGGTGAACCGTCCGAGTCTGGTAG 291 GFP-N_G03.ab1SSGPGESSGSGGEPSE CTCAGGTAGCGAAGGTTCTTCTGGTCCGGGTG SGSSAGTCTTCAGGTTCTGGTGGCGAACCGTCCGAA TCTGGTAGCTCA LCW0401_039_GSGGEPSESGSSGESP 254 GGTTCTGGTGGCGAACCGTCCGAATCTGGTAG 292 GFP-N_H03.ab1GGSSGSESGSGGEPSE CTCAGGTGAATCTCCTGGTGGTTCCAGCGGTTC SGSSCGAGTCAGGTTCTGGTGGCGAACCTTCCGAAT CTGGTAGCTCA LCW0401_040_GSSESGSSEGGPGSGG 255 GGTTCTTCCGAAAGCGGTTCTTCCGAGGGCGG 293 GFP-N_A04.ab1EPSESGSSGSSESGSS TCCAGGTTCCGGTGGTGAACCATCTGAATCTG EGGPGTAGCTCAGGTTCTTCTGAAAGCGGTTCTTCTG AAGGTGGTCCA LCW0401_042_GSEGSSGPGESSGESP 256 GGTAGCGAAGGTTCTTCCGGTCCTGGTGAGTC 294 GFP-N_C04.ab1GGSSGSESGSEGSSGP TTCAGGTGAATCTCCAGGTGGCTCTAGCGGTTC GESSCGAGTCAGGTAGCGAAGGTTCTTCTGGTCCTG GCGAGTCCTCA LCW0401_046_GSSESGSSEGGPGSSE 257 GGTTCCTCTGAAAGCGGTTCTTCCGAAGGCGG 295 GFP-N_D04.ab1SGSSEGGPGSSESGSS TCCAGGTTCTTCCGAAAGCGGTTCTTCTGAGGG EGGPCGGTCCAGGTTCCTCCGAAAGCGGTTCTTCTGA GGGTGGTCCA LCW0401_047_GSGGEPSESGSSGESP 258 GGTTCTGGTGGCGAACCTTCCGAGTCTGGTAG 296 GFP-N_E04.ab1GGSSGSESGESPGGSS CTCAGGTGAATCTCCGGGTGGTTCTAGCGGTTC GSESCGAGTCAGGTGAATCTCCGGGTGGTTCCAGCG GTTCTGAGTCA LCW0401_051_GSGGEPSESGSSGSEG 259 GGTTCTGGTGGCGAACCATCTGAGTCTGGTAG 297 GFP-N_F04.ab1SSGPGESSGESPGGSS CTCAGGTAGCGAAGGTTCTTCCGGTCCAGGCG GSESAGTCTTCAGGTGAATCTCCTGGTGGCTCCAGC GGTTCTGAGTCA LCW0401_053_GESPGGSSGSESGESP 260 GGTGAATCTCCTGGTGGTTCCAGCGGTTCCGA 298 GFP-N_H04.ab1GGSSGSESGESPGGSS GTCAGGTGAATCTCCAGGTGGCTCTAGCGGTT GSESCCGAGTCAGGTGAATCTCCTGGTGGTTCTAGC GGTTCTGAATCA LCW0401_054_GSEGSSGPGESSGSEG 261 GGTAGCGAAGGTTCTTCCGGTCCAGGTGAATC 299 GFP-N_A05.ab1SSGPGESSGSGGEPSE TTCAGGTAGCGAAGGTTCTTCTGGTCCTGGTGA SGSSATCCTCAGGTTCCGGTGGCGAACCATCTGAAT CTGGTAGCTCA LCW0401_059_GSGGEPSESGSSGSEG 262 GGTTCTGGTGGCGAACCATCCGAATCTGGTAG 300 GFP-N_D05.ab1SSGPGESSGESPGGSS CTCAGGTAGCGAAGGTTCTTCTGGTCCTGGCG GSESAATCTTCAGGTGAATCTCCAGGTGGCTCTAGC GGTTCCGAATCA LCW0401_060_GSGGEPSESGSSGSSE 263 GGTTCCGGTGGTGAACCGTCCGAATCTGGTAG 301 GFP-N_E05.ab1SGSSEGGPGSGGEPSE CTCAGGTTCCTCTGAAAGCGGTTCTTCCGAGG SGSSGTGGTCCAGGTTCCGGTGGTGAACCTTCTGAG TCTGGTAGCTCA LCW0401_061_GSSESGSSEGGPGSGG 264 GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGG 302 GFP-N_F05.ab1EPSESGSSGSEGSSGP TCCAGGTTCTGGTGGCGAACCATCTGAATCTG GESSGTAGCTCAGGTAGCGAAGGTTCTTCCGGTCCG GGTGAATCTTCA LCW0401_063_GSGGEPSESGSSGSEG 265 GGTTCTGGTGGTGAACCGTCCGAATCTGGTAG 303 GFP-N_H05.ab1SSGPGESSGSEGSSGP CTCAGGTAGCGAAGGTTCTTCTGGTCCTGGCG GESSAGTCTTCAGGTAGCGAAGGTTCTTCTGGTCCTG GTGAATCTTCA LCW0401_066_GSGGEPSESGSSGSSE 266 GGTTCTGGTGGCGAACCATCCGAGTCTGGTAG 304 GFP-N_B06.ab1SGSSEGGPGSGGEPSE CTCAGGTTCTTCCGAAAGCGGTTCTTCCGAAG SGSSGCGGTCCAGGTTCTGGTGGTGAACCGTCCGAA TCTGGTAGCTCA LCW0401_067_GSGGEPSESGSSGESP 267 GGTTCCGGTGGCGAACCTTCCGAATCTGGTAG 305 GFP-N_C06.ab1GGSSGSESGESPGGSS CTCAGGTGAATCTCCGGGTGGTTCTAGCGGTTC GSESCGAATCAGGTGAATCTCCAGGTGGTTCTAGCG GTTCCGAATCA LCW0401_069_GSGGEPSESGSSGSGG 268 GGTTCCGGTGGTGAACCATCTGAGTCTGGTAG 306 GFP-N_D06.ab1EPSESGSSGESPGGSS CTCAGGTTCCGGTGGCGAACCGTCCGAGTCTG GSESGTAGCTCAGGTGAATCTCCGGGTGGTTCCAGC GGTTCCGAATCA LCW0401_070_GSEGSSGPGESSGSSE 269 GGTAGCGAAGGTTCTTCTGGTCCGGGCGAATC 307 GFP-N_E06.ab1SGSSEGGPGSEGSSGP CTCAGGTTCCTCCGAAAGCGGTTCTTCCGAAG GESSGTGGTCCAGGTAGCGAAGGTTCTTCCGGTCCT GGTGAATCTTCA LCW0401_078_GSSESGSSEGGPGESP 270 GGTTCCTCTGAAAGCGGTTCTTCTGAAGGCGG 308 GFP-N_F06.ab1GGSSGSESGESPGGSS TCCAGGTGAATCTCCGGGTGGCTCCAGCGGTT GSESCTGAATCAGGTGAATCTCCTGGTGGCTCCAGC GGTTCCGAGTCA LCW0401_079_GSEGSSGPGESSGSEG 271 GGTAGCGAAGGTTCTTCTGGTCCAGGCGAGTC 309 GFP-N_G06.ab1SSGPGESSGSGGEPSE TTCAGGTAGCGAAGGTTCTTCCGGTCCTGGCG SGSSAGTCTTCAGGTTCCGGTGGCGAACCGTCCGAA TCTGGTAGCTCA

Example 2: Construction of XTEN_AE36 Segments

A codon library encoding XTEN sequences of 36 amino acid length wasconstructed. The XTEN sequence was designated XTEN_AE36. Its segmentshave the amino acid sequence [X]₃ where X is a 12mer peptide with thesequence: GSPAGSPTSTEE (SEQ ID NO: 6), GSEPATSGSETP (SEQ ID NO: 7),GTSESATPESGP (SEQ ID NO: 8), or GTSTEPSEGSAP (SEQ ID NO: 9). The insertwas obtained by annealing the following pairs of phosphorylatedsynthetic oligonucleotide pairs:

AE1for: (SEQ ID NO: 310) AGGTAGCCCDGCWGGYTCTCCDACYTCYACYGARGA AE1rev:(SEQ ID NO: 311) ACCTTCYTCRGTRGARGTHGGAGARCCWGCHGGGCT AE2for:(SEQ ID NO: 312) AGGTAGCGAACCKGCWACYTCYGGYTCTGARACYCC AE2rev:(SEQ ID NO: 313) ACCTGGRGTYTCAGARCCRGARGTWGCMGGTTCGCT AE3for:(SEQ ID NO: 314) AGGTACYTCTGAAAGCGCWACYCCKGARTCYGGYCC AE3rev:(SEQ ID NO: 315) ACCTGGRCCRGAYTCMGGRGTWGCGCTTTCAGARGT AE4for:(SEQ ID NO: 316) AGGTACYTCTACYGAACCKTCYGARGGYAGCGCWCC AE4rev:(SEQ ID NO: 317) ACCTGGWGCGCTRCCYTCRGAMGGTTCRGTAGARGT

We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor:AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 232) and the non-phosphorylatedoligonucleotide pr_3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 233).The annealed oligonucleotide pairs were ligated, which resulted in amixture of products with varying length that represents the varyingnumber of 12mer repeats ligated to one BbsI/KpnI segment. The productscorresponding to the length of 36 amino acids were isolated from themixture by preparative agarose gel electrophoresis and ligated into theBsaI/KpnI digested stuffer vector pCW0359. Most of the clones in theresulting library designated LCW0402 showed green fluorescence afterinduction which shows that the sequence of XTEN_AE36 had been ligated inframe with the GFP gene and most sequences of XTEN_AE36 show goodexpression.

We screened 96 isolates from library LCW0402 for high level offluorescence by stamping them onto agar plate containing IPTG. The sameisolates were evaluated by PCR and 48 isolates were identified thatcontained segments with 36 amino acids as well as strong fluorescence.These isolates were sequenced and 37 clones were identified thatcontained correct XTEN_AE36 segments. The file names of the nucleotideand amino acid constructs and the sequences for these segments arelisted in Table 12.

TABLE 12 DNA and Amino Acid Sequences for 36-mer motifs SEQ ID SEQ IDFile name Amino acid sequence NO: Nucleotide sequence NO: LCW0402_002GSPAGSPTSTEEGT 318 GGTAGCCCGGCAGGCTCTCCGACCTCTACTGA 355 GFP-N_A07.ab1_SESATPESGPGTSTE GGAAGGTACTTCTGAAAGCGCAACCCCGGAGT PSEGSAPCCGGCCCAGGTACCTCTACCGAACCGTCTGAG GGCAGCGCACCA LCW0402_003_GTSTEPSEGSAPGT 319 GGTACTTCTACCGAACCGTCCGAAGGCAGCGC 356 GFP-N_B07.ab1STEPSEGSAPGTSTE TCCAGGTACCTCTACTGAACCTTCCGAGGGCA PSEGSAPGCGCTCCAGGTACCTCTACCGAACCTTCTGAA GGTAGCGCACCA LCW0402_004_GTSTEPSEGSAPGT 320 GGTACCTCTACCGAACCGTCTGAAGGTAGCGC 357 GFP-N_C07.ab1SESATPESGPGTSES ACCAGGTACCTCTGAAAGCGCAACTCCTGAGT ATPESGPCCGGTCCAGGTACTTCTGAAAGCGCAACCCCG GAGTCTGGCCCA LCW0402_005_GTSTEPSEGSAPGT 321 GGTACTTCTACTGAACCGTCTGAAGGTAGCGC 358 GFP-N_D07.ab1SESATPESGPGTSES ACCAGGTACTTCTGAAAGCGCAACCCCGGAAT ATPESGPCCGGCCCAGGTACCTCTGAAAGCGCAACCCCG GAGTCCGGCCCA LCW0402_006_GSEPATSGSETPGT 322 GGTAGCGAACCGGCAACCTCCGGCTCTGAAAC 359 GFP-N_E07.ab1SESATPESGPGSPA CCCAGGTACCTCTGAAAGCGCTACTCCTGAAT GSPTSTEECCGGCCCAGGTAGCCCGGCAGGTTCTCCGACT TCCACTGAGGAA LCW0402_008_GTSESATPESGPGS 323 GGTACTTCTGAAAGCGCAACCCCTGAATCCGG 360 GFP-N_F07.ab1EPATSGSETPGTSTE TCCAGGTAGCGAACCGGCTACTTCTGGCTCTG PSEGSAPAGACTCCAGGTACTTCTACCGAACCGTCCGAA GGTAGCGCACCA LCW0402_009_GSPAGSPTSTEEGSP 324 GGTAGCCCGGCTGGCTCTCCAACCTCCACTGA 361 GFP-N_G07.ab1AGSPTSTEEGSEPA GGAAGGTAGCCCGGCTGGCTCTCCAACCTCCA TSGSETPCTGAAGAAGGTAGCGAACCGGCTACCTCCGGC TCTGAAACTCCA LCW0402_011_GSPAGSPTSTEEGT 325 GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAG 362 GFP-N_A08.ab1SESATPESGPGTSTE GAAGGTACTTCTGAAAGCGCTACTCCTGAGTC PSEGSAPTGGTCCAGGTACCTCTACTGAACCGTCCGAAG GTAGCGCTCCA LCW0402_012_GSPAGSPTSTEEGSP 326 GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAG 363 GFP-N_B08.ab1AGSPTSTEEGTSTEP GAAGGTAGCCCGGCTGGTTCTCCGACTTCTACT SEGSAPGAGGAAGGTACTTCTACCGAACCTTCCGAAGG TAGCGCTCCA LCW0402_013_ GTSESATPESGPGT327 GGTACTTCTGAAAGCGCTACTCCGGAGTCCGG 364 GFP-N_C08.ab1 STEPSEGSAPGTSTETCCAGGTACCTCTACCGAACCGTCCGAAGGCA PSEGSAPGCGCTCCAGGTACTTCTACTGAACCTTCTGAGG GTAGCGCTCCA LCW0402_014_GTSTEPSEGSAPGSP 328 GGTACCTCTACCGAACCTTCCGAAGGTAGCGC 365 GFP-N_D08.ab1AGSPTSTREGTSTEP TCCAGGTAGCCCGGCAGGTTCTCCTACTTCCAC SEGSAPTGAGGAAGGTACTTCTACCGAACCTTCTGAGG GTAGCGCACCA LCW0402_015_GSEPATSGSETPGSP 329 GGTAGCGAACCGGCTACTTCCGGCTCTGAGAC 366 GFP-N_E08.ab1AGSPTSTREGTSES TCCAGGTAGCCCTGCTGGCTCTCCGACCTCTAC ATPESGPCGAAGAAGGTACCTCTGAAAGCGCTACCCCTG AGTCTGGCCCA LCW0402_016_ GTSTEPSEGSAPGT330 GGTACTTCTACCGAACCTTCCGAGGGCAGCGC 367 GFP-N_F08.ab1 SESATPESGPGTSESACCAGGTACTTCTGAAAGCGCTACCCCTGAGT ATPESGPCCGGCCCAGGTACTTCTGAAAGCGCTACTCCT GAATCCGGTCCA LCW0402_020_GTSTEPSEGSAPGS 331 GGTACTTCTACTGAACCGTCTGAAGGCAGCGC 368 GFP-N_G08.ab1EPATSGSETPGSPA ACCAGGTAGCGAACCGGCTACTTCCGGTTCTG GSPTSTEEAAACCCCAGGTAGCCCAGCAGGTTCTCCAACT TCTACTGAAGAA LCW0402_023_GSPAGSPTSTEEGT 332 GGTAGCCCTGCTGGCTCTCCAACCTCCACCGA 369 GFP-N_A09.ab1SESATPESGPGSEPA AGAAGGTACCTCTGAAAGCGCAACCCCTGAAT TSGSETPCCGGCCCAGGTAGCGAACCGGCAACCTCCGGT TCTGAAACCCCA LCW0402_024_GTSESATPESGPGSP 333 GGTACTTCTGAAAGCGCTACTCCTGAGTCCGG 370 GFP-N_B09.ab1AGSPTSTEEGSPAG CCCAGGTAGCCCGGCTGGCTCTCCGACTTCCA SPTSTEECCGAGGAAGGTAGCCCGGCTGGCTCTCCAACT TCTACTGAAGAA LCW0402_025_GTSTEPSEGSAPGT 334 GGTACCTCTACTGAACCTTCTGAGGGCAGCGC 371 GFP-N_C09.ab1SESATPESGPGTSTE TCCAGGTACTTCTGAAAGCGCTACCCCGGAGT PSEGSAPCCGGTCCAGGTACTTCTACTGAACCGTCCGAA GGTAGCGCACCA LCW0402_026_GSPAGSPTSTEEGT 335 GGTAGCCCGGCAGGCTCTCCGACTTCCACCGA 372 GFP-N_D09.ab1SLEPSEGSAPGSEPA GGAAGGTACCTCTACTGAACCTTCTGAGGGTA TSGSETPGCGCTCCAGGTAGCGAACCGGCAACCTCTGGC TCTGAAACCCCA LCW0402_027_GSPAGSPTSTEEGT 336 GGTAGCCCAGCAGGCTCTCCGACTTCCACTGA 373 GFP-N_E09.ab1STEPSEGSAPGTSTE GGAAGGTACTTCTACTGAACCTTCCGAAGGCA PSEGSAPGCGCACCAGGTACCTCTACTGAACCTTCTGAG GGCAGCGCTCCA LCW0402_032_GSEPATSGSETPGT 337 GGTAGCGAACCTGCTACCTCCGGTTCTGAAAC 374 GFP-N_H09.ab1SESATPESGPGSPA CCCAGGTACCTCTGAAAGCGCAACTCCGGAGT GSPTSTEECTGGTCCAGGTAGCCCTGCAGGTTCTCCTACCT CCACTGAGGAA LCW0402_034_GTSESATPESGPGT 338 GGTACCTCTGAAAGCGCTACTCCGGAGTCTGG 375 GFP-N_A10.ab1STEPSEGSAPGTSTE CCCAGGTACCTCTACTGAACCGTCTGAGGGTA PSEGSAPGCGCTCCAGGTACTTCTACTGAACCGTCCGAA GGTAGCGCACCA LCW0402_036_GSPAGSPTSTEEGT 339 GGTAGCCCGGCTGGTTCTCCGACTTCCACCGA 376 GFP-N_C10.ab1STEPSEGSAPGTSTE GGAAGGTACCTCTACTGAACCTTCTGAGGGTA PSEGSAPGCGCTCCAGGTACCTCTACTGAACCTTCCGAA GGCAGCGCTCCA LCW0402_039_GTSTEPSEGSAPGT 340 GGTACTTCTACCGAACCGTCCGAGGGCAGCGC 377 GFP-N_E10.ab1STEPSEGSAPGTSTE TCCAGGTACTTCTACTGAACCTTCTGAAGGCA PSEGSAPGCGCTCCAGGTACTTCTACTGAACCTTCCGAA GGTAGCGCACCA LCW0402_040_GSEPATSGSETPGT 341 GGTAGCGAACCTGCAACCTCTGGCTCTGAAAC 378 GFP-N_F10.ab1SESATPESGPGTSTE CCCAGGTACCTCTGAAAGCGCTACTCCTGAAT PSEGSAPCTGGCCCAGGTACTTCTACTGAACCGTCCGAG GGCAGCGCACCA LCW0402_041_GTSTEPSEGSAPGSP 342 GGTACTTCTACCGAACCGTCCGAGGGTAGCGC 379 GFP-N_G10.ab1AGSPTSTEEGTSTEP ACCAGGTAGCCCAGCAGGTTCTCCTACCTCCA SEGSAPCCGAGGAAGGTACTTCTACCGAACCGTCCGAG GGTAGCGCACCA LCW0402_050_GSEPATSGSETPGT 343 GGTAGCGAACCGGCAACCTCCGGCTCTGAAAC 380 GFP-N_A11.ab1SESATPESGPGSEPA TCCAGGTACTTCTGAAAGCGCTACTCCGGAAT TSGSETPCCGGCCCAGGTAGCGAACCGGCTACTTCCGGC TCTGAAACCCCA LCW0402_051_GSEPATSGSETPGT 344 GGTAGCGAACCGGCAACTTCCGGCTCTGAAAC 381 GFP-N_B11.ab1SESATPESGPGSEPA CCCAGGTACTTCTGAAAGCGCTACTCCTGAGT TSGSETPCTGGCCCAGGTAGCGAACCTGCTACCTCTGGC TCTGAAACCCCA LCW0402_059_GSEPATSGSETPGS 345 GGTAGCGAACCGGCAACCTCTGGCTCTGAAAC 382 GFP-N_E11.ab1EPATSGSETPGTSTE TCCAGGTAGCGAACCTGCAACCTCCGGCTCTG PSEGSAPAAACCCCAGGTACTTCTACTGAACCTTCTGAG GGCAGCGCACCA LCW0402_060_GTSESATPESGPGS 346 GGTACTTCTGAAAGCGCTACCCCGGAATCTGG 383 GFP-N_F11.ab1EPATSGSETPGSEP CCCAGGTAGCGAACCGGCTACTTCTGGTTCTG ATSGSETPAAACCCCAGGTAGCGAACCGGCTACCTCCGGT TCTGAAACTCCA LCW0402_061_GTSTEPSEGSAPGT 347 GGTACCTCTACTGAACCTTCCGAAGGCAGCGC 384 GFP-N_G11.ab1STEPSEGSAPGTSES TCCAGGTACCTCTACCGAACCGTCCGAGGGCA ATPESGPGCGCACCAGGTACTTCTGAAAGCGCAACCCCT GAATCCGGTCCA LCW0402_065_GSEPATSGSETPGT 348 GGTAGCGAACCGGCAACCTCTGGCTCTGAAAC 385 GFP-N_A12.ab1SESATPESGPGTSES CCCAGGTACCTCTGAAAGCGCTACTCCGGAAT ATPESGPCTGGTCCAGGTACTTCTGAAAGCGCTACTCCG GAATCCGGTCCA LCW0402_066_GSEPATSGSETPGS 349 GGTAGCGAACCTGCTACCTCCGGCTCTGAAAC 386 GFP-N_B12.ab1EPATSGSETPGTSTE TCCAGGTAGCGAACCGGCTACTTCCGGTTCTG PSEGSAPAAACTCCAGGTACCTCTACCGAACCTTCCGAA GGCAGCGCACCA LCW0402_067_GSEPATSGSETPGT 350 GGTAGCGAACCTGCTACTTCTGGTTCTGAAACT 387 GFP-N_C12.ab1STEPSEGSAPGSEPA CCAGGTACTTCTACCGAACCGTCCGAGGGTAG TSGSETPCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTC TGAAACTCCA LCW0402_069_ GTSTEPSEGSAPGT351 GGTACCTCTACCGAACCGTCCGAGGGTAGCGC 388 GFP-N_D12.ab1 STEPSEGSAPGSEPAACCAGGTACCTCTACTGAACCGTCTGAGGGTA TSGSETPGCGCTCCAGGTAGCGAACCGGCAACCTCCGGT TCTGAAACTCCA LCW0402_073_GTSTEPSEGSAPGS 352 GGTACTTCTACTGAACCTTCCGAAGGTAGCGC 389 GFP-N_F12.ab1EPATSGSETPGSPA TCCAGGTAGCGAACCTGCTACTTCTGGTTCTGA GSPTSTEEAACCCCAGGTAGCCCGGCTGGCTCTCCGACCT CCACCGAGGAA LCW0402_074_GSEPATSGSETPGSP 353 GGTAGCGAACCGGCTACTTCCGGCTCTGAGAC 390 GFP-N_G12.ab1AGSPTSTEEGTSES TCCAGGTAGCCCAGCTGGTTCTCCAACCTCTAC ATPESGPTGAGGAAGGTACTTCTGAAAGCGCTACCCCTG AATCTGGTCCA LCW0402_075_ GTSESATPESGPGS354 GGTACCTCTGAAAGCGCAACTCCTGAGTCTGG 391 GFP-N_H12.ab1 EPATSGSETPGTSESCCCAGGTAGCGAACCTGCTACCTCCGGCTCTG ATPESGPAGACTCCAGGTACCTCTGAAAGCGCAACCCCG GAATCTGGTCCA

Example 3: Construction of XTEN_AF36 Segments

A codon library encoding sequences of 36 amino acid length wasconstructed. The sequences were designated XTEN_AF36. Its segments havethe amino acid sequence [X]₃ where X is a 12mer peptide with thesequence: GSTSESPSGTAP (SEQ ID NO: 10), GTSTPESGSASP (SEQ ID NO: 11),GTSPSGESSTAP (SEQ ID NO: 12), or GSTSSTAESPGP (SEQ ID NO: 13). Theinsert was obtained by annealing the following pairs of phosphorylatedsynthetic oligonucleotide pairs:

(SEQ ID NO: 392) AF1for: AGGTTCTACYAGCGAATCYCCKTCTGGYACYGCWCC(SEQ ID NO: 393) AF1rev: ACCTGGWGCRGTRCCAGAMGGRGATTCGCTRGTAGA(SEQ ID NO: 394) AF2for: AGGTACYTCTACYCCKGAAAGCGGYTCYGCWTCTCC(SEQ ID NO: 395) AF2rev: ACCTGGAGAWGCRGARCCGCTTTCMGGRGTAGARGT(SEQ ID NO: 396) AF3for: AGGTACYTCYCCKAGCGGYGAATCTTCTACYGCWCC(SEQ ID NO: 397) AF3rev: ACCTGGWGCRGTAGAAGATTCRCCGCTMGGRGARGT(SEQ ID NO: 398) AF4for: AGGTTCYACYAGCTCTACYGCWGAATCTCCKGGYCC(SEQ ID NO: 399) AF4rev: ACCTGGRCCMGGAGATTCWGCRGTAGAGCTRGTRGA

We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor:AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 232) and the non-phosphorylatedoligonucleotide pr_3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 233).The annealed oligonucleotide pairs were ligated, which resulted in amixture of products with varying length that represents the varyingnumber of 12mer repeats ligated to one BbsI/KpnI segment The productscorresponding to the length of 36 amino acids were isolated from themixture by preparative agarose gel electrophoresis and ligated into theBsaI/KpnI digested stuffer vector pCW0359. Most of the clones in theresulting library designated LCW0403 showed green fluorescence afterinduction which shows that the sequence of XTEN_AF36 had been ligated inframe with the GFP gene and most sequences of XTEN_AF36 show goodexpression.

We screened 96 isolates from library LCW0403 for high level offluorescence by stamping them onto agar plate containing IPTG. The sameisolates were evaluated by PCR and 48 isolates were identified thatcontained segments with 36 amino acids as well as strong fluorescence.These isolates were sequenced and 44 clones were identified thatcontained correct XTEN_AF36 segments. The file names of the nucleotideand amino acid constructs and the sequences for these segments arelisted in Table 13.

TABLE 13 DNA and Amino Acid Sequences for 36-mer motifs SEQ SEQAmino acid ID ID File name sequence NO: Nucleotide sequence NO:LCW0403_004_ GTSTPESGSASPGTS 400 GGTACTTCTACTCCGGAAAGCGGTTCCGCATCT 444GFP-N_A01.ab1 PSGESSTAPGTSPSG CCAGGTACTTCTCCTAGCGGTGAATCTTCTACT ESSTAPGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCT ACTGCTCCA LCW0403_005_ GTSPSGESSTAPGST401 GGTACTTCTCCGAGCGGTGAATCTTCTACCGCA 445 GFP-N_B01.ab1 SSTAESPGPGTSPSGCCAGGTTCTACTAGCTCTACCGCTGAATCTCCG ESSTAPGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCT ACTGCTCCA LCW0403_006_ GSTSSTAESPGPGTS402 GGTTCCACCAGCTCTACTGCTGAATCTCCTGGT 446 GFP-N_C01.ab1 PSGESSTAPGTSTPESCCAGGTACCTCTCCTAGCGGTGAATCTTCTACT GSASPGCTCCAGGTACTTCTACTCCTGAAAGCGGCTCT GCTTCTCCA LCW0403_007_ GSTSSTAESPGPGST403 GGTTCTACCAGCTCTACTGCAGAATCTCCTGGC 447 GFP-N_D01.ab1 SSTAESPGPGTSPSGCCAGGTTCCACCAGCTCTACCGCAGAATCTCCG ESSTAPGGTCCAGGTACTTCCCCTAGCGGTGAATCTTCT ACCGCACCA LCW0403_008_ GSTSSTAESPGPGTS404 GGTTCTACTAGCTCTACTGCTGAATCTCCTGGCC 448 GFP-N_E01.ab1PSGESSTAPGTSTPES CAGGTACTTCTCCTAGCGGTGAATCTTCTACCG GSASPCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTG CATCTCCA LCW0403_010_ GSTSSTAESPGPGTS405 GGTTCTACCAGCTCTACCGCAGAATCTCCTGGT 449 GFP-N_F01.ab1 TPESGSASPGSTSESPCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCA SGTAPTCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCA CTGCACCA LCW0403_011_ GSTSSTAESPGPGTS406 GGTTCTACTAGCTCTACTGCAGAATCTCCTGGC 450 GFP-N_G01.ab1 TPESGSASPGTSTPESCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCA GSASPTCTCCAGGTACTTCTACCCCTGAAAGCGGTTCT GCATCTCCA LCW0403_012_ GSTSESPSGTAPGTS407 GGTTCTACCAGCGAATCTCCTTCTGGCACCGCT 451 GFP-N_H01.ab1 PSGESSTAPGSTSESPCCAGGTACCTCTCCTAGCGGCGAATCTTCTACC SGTAPGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGC ACTGCACCA LCW0403_013_ GSTSSTAESPGPGST408 GGTTCCACCAGCTCTACTGCAGAATCTCCGGGC 452 GFP-N_A02.ab1 SSTAESPGPGTSPSGCCAGGTTCTACTAGCTCTACTGCAGAATCTCCG ESSTAPGGTCCAGGTACTTCTCCTAGCGGCGAATCTTCT ACCGCTCCA LCW0403_014_ GSTSSTAESPGPGTS409 GGTTCCACTAGCTCTACTGCAGAATCTCCTGGC 453 GFP-N_B02.ab1 TPESGSASPGSTSESPCCAGGTACCTCTACCCCTGAAAGCGGCTCTGCA SGTAPTCTCCAGGTTCTACCAGCGAATCCCCGTCTGGC ACCGCACCA LCW0403_015_ GSTSSTAESPGPGST410 GGTTCTACTAGCTCTACTGCTGAATCTCCGGGT 454 GFP-N_C02.ab1 SSTAESPGPGTSPSGCCAGGTTCTACCAGCTCTACTGCTGAATCTCCT ESSTAPGGTCCAGGTACCTCCCCGAGCGGTGAATCTTCT ACTGCACCA LCW0403_017_ GSTSSTAESPGPGST411 GGTTCTACCAGCTCTACCGCTGAATCTCCTGGC 455 GFP-N_D02.ab1 SESPSGTAPGSTSSTCCAGGTTCTACCAGCGAATCCCCGTCTGGCACC AESPGPGCACCAGGTTCTACTAGCTCTACCGCTGAATCT CCGGGTCCA LCW0403_018_ GSTSSTAESPGPGST412 GGTTCTACCAGCTCTACCGCAGAATCTCCTGGC 456 GFP-N_E02.ab1 SSTAESPGPGSTSSTCCAGGTTCCACTAGCTCTACCGCTGAATCTCCT AESPGPGGTCCAGGTTCTACTAGCTCTACCGCTGAATCT CCTGGTCCA LCW0403_019_ GSTSESPSGTAPGST413 GGTTCTACTAGCGAATCCCCTTCTGGTACTGCTC 457 GFP-N_F02.ab1 SSTAESPGPGSTSSTCAGGTTCCACTAGCTCTACCGCTGAATCTCCTG AESPGPGCCCAGGTTCCACTAGCTCTACTGCAGAATCTC CTGGTCCA LCW0403_023_ GSTSESPSGTAPGST414 GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTC 458 GFP-N_H02.ab1SESPSGTAPGSTSESP CAGGTTCTACCAGCGAATCCCCGTCTGGTACTG SGTAPCTCCAGGTTCTACCAGCGAATCTCCTTCTGGTA CTGCACCA LCW0403_024_ GSTSSTAESPGPGST415 GGTTCCACCAGCTCTACTGCTGAATCTCCTGGC 459 GFP-N_A03.ab1 SSTAESPGPGSTSSTCCAGGTTCTACCAGCTCTACTGCTGAATCTCCG AESPGPGGCCCAGGTTCCACCAGCTCTACCGCTGAATCT CCGGGTCCA LCW0403_025_ GSTSSTAESPGPGST416 GGTTCCACTAGCTCTACCGCAGAATCTCCTGGT 460 GFP-N_B03.ab1 SSTAESPGPGTSPSGCCAGGTTCTACTAGCTCTACTGCTGAATCTCCG ESSTAPGGTCCAGGTACCTCCCCTAGCGGCGAATCTTCT ACCGCTCCA LCW0403_028_ GSSPSASTGTGPGSS417 GGTTCTAGCCCTTCTGCTTCCACCGGTACCGGC 461 GFP-N_D03.ab1 TPSGATGSPGSSTPSCCAGGTAGCTCTACTCCGTCTGGTGCAACTGGC GATGSPTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACC GGCTCCCCA LCW0403_029_ GTSPSGESSTAPGTS418 GGTACTTCCCCTAGCGGTGAATCTTCTACTGCTC 462 GFP-N_E03.ab1 TPESGSASPGSTSSTCAGGTACCTCTACTCCGGAAAGCGGCTCCGCAT AESPGPCTCCAGGTTCTACTAGCTCTACTGCTGAATCTCC TGGTCCA LCW0403_030_ GSTSSTAESPGPGST419 GGTTCTACTAGCTCTACCGCTGAATCTCCGGGT 463 GFP-N_F03.ab1 SSTAESPGPGTSTPESCCAGGTTCTACCAGCTCTACTGCAGAATCTCCT GSASPGGCCCAGGTACTTCTACTCCGGAAAGCGGTTCC GCTTCTCCA LCW0403_031_ GTSPSGESSTAPGST420 GGTACTTCTCCTAGCGGTGAATCTTCTACCGCTC 464 GFP-N_G03.ab1SSTAESPGPGTSTPES CAGGTTCTACCAGCTCTACTGCTGAATCTCCTG GSASPGCCCAGGTACTTCTACCCCGGAAAGCGGCTCCG CTTCTCCA LCW0403_033_ GSTSESPSGTAPGST421 GGTTCTACTAGCGAATCCCCTTCTGGTACTGCA 465 GFP-N_H03.ab1 SSTAESPGPGSTSSTCCAGGTTCTACCAGCTCTACTGCTGAATCTCCG AESPGPGGCCCAGGTTCCACCAGCTCTACCGCAGAATCT CCTGGTCCA LCW0403_035_ GSTSSTAESPGPGST422 GGTTCCACCAGCTCTACCGCTGAATCTCCGGGC 466 GFP-N_A04.ab1 SESPSGTAPGSTSSTCCAGGTTCTACCAGCGAATCCCCTTCTGGCACT AESPGPGCACCAGGTTCTACTAGCTCTACCGCAGAATCT CCGGGCCCA LCW0403_036_ GSTSSTAESPGPGTS423 GGTTCTACCAGCTCTACTGCTGAATCTCCGGGT 467 GFP-N_B04.ab1 PSGESSTAPGTSTPESCCAGGTACTTCCCCGAGCGGTGAATCTTCTACT GSASPGCACCAGGTACTTCTACTCCGGAAAGCGGTTCC GCTTCTCCA LCW0403_039_ GSTSESPSGTAPGST424 GGTTCTACCAGCGAATCTCCTTCTGGCACCGCT 468 GFP-N_C04.ab1 SESPSGTAPGTSPSGCCAGGTTCTACTAGCGAATCCCCGTCTGGTACC ESSTAPGCACCAGGTACTTCTCCTAGCGGCGAATCTTCT ACCGCACCA LCW0403_041_ GSTSESPSGTAPGST425 GGTTCTACCAGCGAATCCCCTTCTGGTACTGCT 469 GFP-N_D04.ab1 SESPSGTAPGTSTPESCCAGGTTCTACCAGCGAATCCCCTTCTGGCACC GSASPGCACCAGGTACTTCTACCCCTGAAAGCGGCTCC GCTTCTCCA LCW0403_044_ GTSTPESGSASPGST426 GGTACCTCTACTCCTGAAAGCGGTTCTGCATCT 470 GFP-N_E04.ab1 SSTAESPGPGSTSSTCCAGGTTCCACTAGCTCTACCGCAGAATCTCCG AESPGPGGCCCAGGTTCTACTAGCTCTACTGCTGAATCT CCTGGCCCA LCW0403_046_ GSTSESPSGTAPGST427 GGTTCTACCAGCGAATCCCCTTCTGGCACTGCA 471 GFP-N_F04.ab1 SESPSGTAPGTSPSGCCAGGTTCTACTAGCGAATCCCCTTCTGGTACC ESSTAPGCACCAGGTACTTCTCCGAGCGGCGAATCTTCT ACTGCTCCA LCW0403_047_ GSTSSTAESPGPGST428 GGTTCTACTAGCTCTACCGCTGAATCTCCTGGC 472 GFP-N_G04.ab1 SSTAESPGPGSTSESPCCAGGTTCCACTAGCTCTACCGCAGAATCTCCG SGTAPGGCCCAGGTTCTACTAGCGAATCCCCTTCTGGT ACCGCTCCA LCW0403_049_ GSTSSTAESPGPGST429 GGTTCCACCAGCTCTACTGCAGAATCTCCTGGC 473 GFP-N_H04.ab1 SSTAESPGPGTSTPESCCAGGTTCTACTAGCTCTACCGCAGAATCTCCT GSASPGGTCCAGGTACCTCTACTCCTGAAAGCGGTTCC GCATCTCCA LCW0403_051_ GSTSSTAESPGPGST430 GGTTCTACTAGCTCTACTGCTGAATCTCCGGGC 474 GFP-N_A05.ab1 SSTAESPGPGSTSESPCCAGGTTCTACTAGCTCTACCGCTGAATCTCCG SGTAPGGTCCAGGTTCTACTAGCGAATCTCCTTCTGGT ACCGCTCCA LCW0403_053_ GTSPSGESSTAPGST431 GGTACCTCCCCGAGCGGTGAATCTTCTACTGCA 475 GFP-N_B05.ab1 SESPSGTAPGSTSSTCCAGGTTCTACTAGCGAATCCCCTTCTGGTACT AESPGPGCTCCAGGTTCCACCAGCTCTACTGCAGAATCT CCGGGTCCA LCW0403_054_ GSTSESPSGTAPGTS432 GGTTCTACTAGCGAATCCCCGTCTGGTACTGCT 476 GFP-N_C05.ab1 PSGESSTAPGSTSSTCCAGGTACTTCCCCTAGCGGTGAATCTTCTACT AESPGPGCTCCAGGTTCTACCAGCTCTACCGCAGAATCT CCGGGTCCA LCW0403_057_ GSTSSTAESPGPGST433 GGTTCTACCAGCTCTACCGCTGAATCTCCTGGC 477 GFP-N_D05.ab1 SESPSGTAPGTSPSGCCAGGTTCTACTAGCGAATCTCCGTCTGGCACC ESSTAPGCACCAGGTACTTCCCCTAGCGGTGAATCTTCT ACTGCACCA LCW0403_058_ GSTSESPSGTAPGST434 GGTTCTACTAGCGAATCTCCTTCTGGCACTGCA 478 GFP-N_E05.ab1 SESPSGTAPGTSTPESCCAGGTTCTACCAGCGAATCTCCGTCTGGCACT GSASPGCACCAGGTACCTCTACCCCTGAAAGCGGTTCC GCTTCTCCA LCW0403_060_ GTSTPESGSASPGST435 GGTACCTCTACTCCGGAAAGCGGTTCCGCATCT 479 GFP-N_F05.ab1 SESPSGTAPGSTSSTCCAGGTTCTACCAGCGAATCCCCGTCTGGCACC AESPGPGCACCAGGTTCTACTAGCTCTACTGCTGAATCT CCGGGCCCA LCW0403_063_ GSTSSTAESPGPGTS436 GGTTCTACTAGCTCTACTGCAGAATCTCCGGGC 480 GFP-N_G05.ab1 PSGESSTAPGTSPSGCCAGGTACCTCTCCTAGCGGTGAATCTTCTACC ESSTAPGCTCCAGGTACTTCTCCGAGCGGTGAATCTTCT ACCGCTCCA LCW0403_064_ GTSPSGESSTAPGTS437 GGTACCTCCCCTAGCGGCGAATCTTCTACTGCT 481 GFP-N_H05.ab1 PSGESSTAPGTSPSGCCAGGTACCTCTCCTAGCGGCGAATCTTCTACC ESSTAPGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCT ACCGCACCA LCW0403_065_ GSTSSTAESPGPGTS438 GGTTCCACTAGCTCTACTGCTGAATCTCCTGGC 482 GFP-N_A06.ab1 TPESGSASPGSTSESPCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCT SGTAPTCTCCAGGTTCTACTAGCGAATCTCCGTCTGGC ACCGCACCA LCW0403_066_ GSTSESPSGTAPGTS439 GGTTCTACTAGCGAATCTCCGTCTGGCACTGCT 483 GFP-N_B06.ab1 PSGESSTAPGTSPSGCCAGGTACTTCTCCTAGCGGTGAATCTTCTACC ESSTAPGCTCCAGGTACTTCCCCTAGCGGCGAATCTTCT ACCGCTCCA LCW0403_067_ GSTSESPSGTAPGTS440 GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTC 484 GFP-N_C06.ab1 TPESGSASPGSTSSTCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTT AESPGPCTCCAGGTTCCACTAGCTCTACCGCTGAATCTC CGGGTCCA LCW0403_068_ GSTSSTAESPGPGST441 GGTTCCACTAGCTCTACTGCTGAATCTCCTGGC 485 GFP-N_D06.ab1 SSTAESPGPGSTSESPCCAGGTTCTACCAGCTCTACCGCTGAATCTCCT SGTAPGGCCCAGGTTCTACCAGCGAATCTCCGTCTGGC ACCGCACCA LCW0403_069_ GSTSESPSGTAPGTS442 GGTTCTACTAGCGAATCCCCGTCTGGTACCGCA 486 GFP-N_E06.ab1 TPESGSASPGTSTPESCCAGGTACTTCTACCCCGGAAAGCGGCTCTGCT GSASPTCTCCAGGTACTTCTACCCCGGAAAGCGGCTCC GCATCTCCA LCW0403_070_ GSTSESPSGTAPGTS443 GGTTCTACTAGCGAATCCCCGTCTGGTACTGCT 487 GFP-N_F06.ab1 TPESGSASPGTSTPESCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCT GSASPTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCT GCATCTCCA

Example 4: Construction of XTEN_AG36 Segments

A codon library encoding sequences of 36 amino acid length wasconstructed. The sequences were designated XTEN_AG36. Its segments havethe amino acid sequence [X]₃ where X is a 12mer peptide with thesequence: GTPGSGTASSSP (SEQ ID NO: 14), GSSTPSGATGSP (SEQ ID NO: 15),GSSPSASTGTGP (SEQ ID NO: 16), or GASPGTSSTGSP (SEQ ID NO: 17). Theinsert was obtained by annealing the following pairs of phosphorylatedsynthetic oligonucleotide pairs:

(SEQ ID NO: 488) AG1for: AGGTACYCCKGGYAGCGGTACYGCWTCTTCYTCTCC(SEQ ID NO: 489) AG1rev: ACCTGGAGARGAAGAWGCRGTACCGCTRCCMGGRGT(SEQ ID NO: 490) AG2for: AGGTAGCTCTACYCCKTCTGGTGCWACYGGYTCYCC(SEQ ID NO: 491) AG2rev: ACCTGGRGARCCRGTWGCACCAGAMGGRGTAGAGCT(SEQ ID NO: 492) AG3for: AGGTTCTAGCCCKTCTGCWTCYACYGGTACYGGYCC(SEQ ID NO: 493) AG3rev: ACCTGGRCCRGTACCRGTRGAWGCAGAMGGGCTAGA(SEQ ID NO: 494) AG4for: AGGTGCWTCYCCKGGYACYAGCTCTACYGGTTCTCC(SEQ ID NO: 495) AG4rev: ACCTGGAGAACCRGTAGAGCTRGTRCCMGGRGAWGC

We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor:AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 232) and the non-phosphorylatedoligonucleotide pr_3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 233).The annealed oligonucleotide pairs were ligated, which resulted in amixture of products with varying length that represents the varyingnumber of 12mer repeats ligated to one BbsI/KpnI segment. The productscorresponding to the length of 36 amino acids were isolated from themixture by preparative agarose gel electrophoresis and ligated into theBsaI/KpnI digested stuffer vector pCW0359. Most of the clones in theresulting library designated LCW0404 showed green fluorescence afterinduction which shows that the sequence of XTEN_AG36 had been ligated inframe with the GFP gene and most sequences of XTEN_AG36 show goodexpression.

We screened 96 isolates from library LCW0404 for high level offluorescence by stamping them onto agar plate containing IPTG. The sameisolates were evaluated by PCR and 48 isolates were identified thatcontained segments with 36 amino acids as well as strong fluorescence.These isolates were sequenced and 44 clones were identified thatcontained correct XTEN_AG36 segments. The file names of the nucleotideand amino acid constructs and the sequences for these segments arelisted in Table 14.

TABLE 14 DNA and Amino Acid Sequences for 36-mer motifs SEQ SEQAmino acid ID ID File name sequence NO: Nucleotide sequence NO:LCW0404_001_ GASPGTSSTGSPGTPG 496 GGTGCATCCCCGGGCACTAGCTCTACCGGTT 540GFP-N_A07.ab1 SGTASSSPGSSTPSGA CTCCAGGTACTCCTGGTAGCGGTACTGCTTC TGSPTTCTTCTCCAGGTAGCTCTACTCCTTCTGGTG CTACTGGTTCTCCA LCW0404_003_GSSTPSGATGSPGSSP 497 GGTAGCTCTACCCCTTCTGGTGCTACCGGCT 541 GFP-N_B07.ab1SASTGTGPGSSTPSGA CTCCAGGTTCTAGCCCGTCTGCTTCTACCGGT TGSPACCGGTCCAGGTAGCTCTACCCCTTCTGGTG CTACTGGTTCTCCA LCW0404_006_GASPGTSSTGSPGSSP 498 GGTGCATCTCCGGGTACTAGCTCTACCGGTT 542 GFP-N_C07.ab1SASTGTGPGSSTPSGA CTCCAGGTTCTAGCCCTTCTGCTTCCACTGGT TGSPACCGGCCCAGGTAGCTCTACCCCGTCTGGTG CTACTGGTTCCCCA LCW0404_007_GTPGSGTASSSPGSST 499 GGTACTCCGGGCAGCGGTACTGCTTCTTCCT 543 GFP-N_D07.ab1PSGATGSPGASPGTSS CTCCAGGTAGCTCTACCCCTTCTGGTGCAAC TGSPTGGTTCCCCAGGTGCATCCCCTGGTACTAGC TCTACCGGTTCTCCA LCW0404_009_GTPGSGTASSSPGASP 500 GGTACCCCTGGCAGCGGTACTGCTTCTTCTTC 544 GFP-N_E07.ab1GTSSTGSPGSRPSAST TCCAGGTGCTTCCCCTGGTACCAGCTCTACC GTGPGGTTCTCCAGGTTCTAGACCTTCTGCATCCAC CGGTACTGGTCCA LCW0404_011_GASPGTSSTGSPGSST 501 GGTGCATCTCCTGGTACCAGCTCTACCGGTT 545 GFP-N_F07.ab1PSGATGSPGASPGTSS CTCCAGGTAGCTCTACTCCTTCTGGTGCTACT TGSPGGCTCTCCAGGTGCTTCCCCGGGTACCAGCT CTACCGGTTCTCCA LCW0404_012_GTPGSGTASSSPGSST 502 GGTACCCCGGGCAGCGGTACCGCATCTTCCT 546 GFP-N_G07.ab1PSGATGSPGSSTPSGA CTCCAGGTAGCTCTACCCCGTCTGGTGCTAC TGSPCGGTTCCCCAGGTAGCTCTACCCCGTCTGGT GCAACCGGCTCCCCA LCW0404_014_GASPGTSSTGSPGASP 503 GGTGCATCTCCGGGCACTAGCTCTACTGGTT 547 GFP-N_H07.ab1GTSSTGSPGASPGTSS CTCCAGGTGCATCCCCTGGCACTAGCTCTAC TGSPTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCT CTACTGGTTCTCCA LCW0404_015_GSSTPSGATGSPGSSP 504 GGTAGCTCTACTCCGTCTGGTGCAACCGGCT 548 GFP-N_A08.ab1SASTGTGPGASPGTSS CCCCAGGTTCTAGCCCGTCTGCTTCCACTGGT TGSPACTGGCCCAGGTGCTTCCCCGGGCACCAGCT CTACTGGTTCTCCA LCW0404_016_GSSTPSGATGSPGSST 505 GGTAGCTCTACTCCTTCTGGTGCTACCGGTTC 549 GFP-N_B08.ab1PSGATGSPGTPGSGT CCCAGGTAGCTCTACTCCTTCTGGTGCTACTG ASSSPGTTCCCCAGGTACTCCGGGCAGCGGTACTGC TTCTTCCTCTCCA LCW0404_017_GSSTPSGATGSPGSST 506 GGTAGCTCTACTCCGTCTGGTGCAACCGGTT 550 GFP-N_C08.ab1PSGATGSPGASPGTSS CCCCAGGTAGCTCTACTCCTTCTGGTGCTACT TGSPGGCTCCCCAGGTGCATCCCCTGGCACCAGCT CTACCGGTTCTCCA LCW0404_018_GTPGSGTASSSPGSSP 507 GGTACTCCTGGTAGCGGTACCGCATCTTCCT 551 GFP-N_D08.ab1SASTGTGPGSSTPSGA CTCCAGGTTCTAGCCCTTCTGCATCTACCGGT TGSPACCGGTCCAGGTAGCTCTACTCCTTCTGGTG CTACTGGCTCTCCA LCW0404_023_GASPGTSSTGSPGSSP 508 GGTGCTTCCCCGGGCACTAGCTCTACCGGTT 552 GFP-N_F08.ab1SASTGTGPGTPGSGT CTCCAGGTTCTAGCCCTTCTGCATCTACTGGT ASSSPACTGGCCCAGGTACTCCGGGCAGCGGTACTG CTTCTTCCTCTCCA LCW0404_025_GSSTPSGATGSPGSST 509 GGTAGCTCTACTCCGTCTGGTGCTACCGGCT 553 GFP-N_G08.ab1PSGATGSPGASPGTSS CTCCAGGTAGCTCTACCCCTTCTGGTGCAAC TGSPCGGCTCCCCAGGTGCTTCTCCGGGTACCAGC TCTACTGGTTCTCCA LCW0404_029_GTPGSGTASSSPGSST 510 GGTACCCCTGGCAGCGGTACCGCTTCTTCCT 554 GFP-N_A09.ab1PSGATGSPGSSPSAST CTCCAGGTAGCTCTACCCCGTCTGGTGCTAC GTGPTGGCTCTCCAGGTTCTAGCCCGTCTGCATCTA CCGGTACCGGCCCA LCW0404_030_GSSTPSGATGSPGTPG 511 GGTAGCTCTACTCCTTCTGGTGCAACCGGCT 555 GFP-N_B09.ab1SGTASSSPGTPGSGTA CCCCAGGTACCCCGGGCAGCGGTACCGCATC SSSPTTCCTCTCCAGGTACTCCGGGTAGCGGTACT GCTTCTTCTTCTCCA LCW0404_031_GTPGSGTASSSPGSST 512 GGTACCCCGGGTAGCGGTACTGCTTCTTCCT 556 GFP-N_C09.ab1PSGATGSPGASPGTSS CTCCAGGTAGCTCTACCCCTTCTGGTGCAAC TGSPCGGCTCTCCAGGTGCTTCTCCGGGCACCAGC TCTACCGGTTCTCCA LCW0404_034_GSSTPSGATGSPGSST 513 GGTAGCTCTACCCCGTCTGGTGCTACCGGCT 557 GFP-N_D09.ab1PSGATGSPGASPGTSS CTCCAGGTAGCTCTACCCCGTCTGGTGCAAC TGSPCGGCTCCCCAGGTGCATCCCCGGGTACTAGC TCTACCGGTTCTCCA LCW0404_035_GASPGTSSTGSPGTPG 514 GGTGCTTCTCCGGGCACCAGCTCTACTGGTT 558 GFP-N_E09.ab1SGTASSSPGSSTPSGA CTCCAGGTACCCCGGGCAGCGGTACCGCATC TGSPTTCTTCTCCAGGTAGCTCTACTCCTTCTGGTG CAACTGGTTCTCCA LCW0404_036_GSSPSASTGTGPGSST 515 GGTTCTAGCCCGTCTGCTTCCACCGGTACTG 559 GFP-N_F09.ab1PSGATGSPGTPGSGT GCCCAGGTAGCTCTACCCCGTCTGGTGCAAC ASSSPTGGTTCCCCAGGTACCCCTGGTAGCGGTACC GCTTCTTCTTCTCCA LCW0404_037_GASPGTSSTGSPGSSP 516 GGTGCTTCTCCGGGCACCAGCTCTACTGGTT 560 GFP-N_G09.ab1SASTGTGPGSSTPSGA CTCCAGGTTCTAGCCCTTCTGCATCCACCGGT TGSPACCGGTCCAGGTAGCTCTACCCCTTCTGGTG CAACCGGCTCTCCA LCW0404_040_GASPGTSSTGSPGSST 517 GGTGCATCCCCGGGCACCAGCTCTACCGGTT 561 GFP-N_H09.ab1PSGATGSPGSSTPSGA CTCCAGGTAGCTCTACCCCGTCTGGTGCTAC TGSPCGGCTCTCCAGGTAGCTCTACCCCGTCTGGT GCTACTGGCTCTCCA LCW0404_041_GTPGSGTASSSPGSST 518 GGTACCCCTGGTAGCGGTACTGCTTCTTCCTC 562 GFP-N_A10.ab1PSGATGSPGTPGSGT TCCAGGTAGCTCTACTCCGTCTGGTGCTACC ASSSPGGTTCTCCAGGTACCCCGGGTAGCGGTACCG CATCTTCTTCTCCA LCW0404_043_GSSPSASTGTGPGSST 519 GGTTCTAGCCCTTCTGCTTCCACCGGTACTGG 563 GFP-N_C10.ab1PSGATGSPGSSTPSGA CCCAGGTAGCTCTACCCCTTCTGGTGCTACC TGSPGGCTCCCCAGGTAGCTCTACTCCTTCTGGTG CAACTGGCTCTCCA LCW0404_045_GASPGTSSTGSPGSSP 520 GGTGCTTCTCCTGGCACCAGCTCTACTGGTTC 564 GFP-N_D10.ab1SASTGTGPGSSPSAST TCCAGGTTCTAGCCCTTCTGCTTCTACCGGTA GTGPCTGGTCCAGGTTCTAGCCCTTCTGCATCCACT GGTACTGGTCCA LCW0404_047_GTPGSGTASSSPGASP 521 GGTACTCCTGGCAGCGGTACCGCTTCTTCTTC 565 GFP-N_F10.ab1GTSSTGSPGASPGTSS TCCAGGTGCTTCTCCTGGTACTAGCTCTACTG TGSPGTTCTCCAGGTGCTTCTCCGGGCACTAGCTCT ACTGGTTCTCCA LCW0404_048_GSSTPSGATGSPGASP 522 GGTAGCTCTACCCCGTCTGGTGCTACCGGTT 566 GFP-N_G10.ab1GTSSTGSPGSSTPSGA CCCCAGGTGCTTCTCCTGGTACTAGCTCTACC TGSPGGTTCTCCAGGTAGCTCTACCCCGTCTGGTG CTACTGGCTCTCCA LCW0404_049_GSSTPSGATGSPGTPG 523 GGTAGCTCTACCCCGTCTGGTGCTACTGGTT 567 GFP-N_H10.ab1SGTASSSPGSSTPSGA CTCCAGGTACTCCGGGCAGCGGTACTGCTTC TGSPTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTG CTACTGGCTCTCCA LCW0404_050_GASPGTSSTGSPGSSP 524 GGTGCATCTCCTGGTACCAGCTCTACTGGTT 568 GFP-N_A11.ab1SASTGTGPGSSTPSGA CTCCAGGTTCTAGCCCTTCTGCTTCTACCGGT TGSPACCGGTCCAGGTAGCTCTACTCCTTCTGGTG CTACCGGTTCTCCA LCW0404_051_GSSTPSGATGSPGSST 525 GGTAGCTCTACCCCGTCTGGTGCTACTGGCT 569 GFP-N_B11.ab1PSGATGSPGSSTPSGA CTCCAGGTAGCTCTACTCCTTCTGGTGCTACT TGSPGGTTCCCCAGGTAGCTCTACCCCGTCTGGTG CAACTGGCTCTCCA LCW0404_052_GASPGTSSTGSPGTPG 526 GGTGCATCCCCGGGTACCAGCTCTACCGGTT 570 GFP-N_C11.ab1SGTASSSPGASPGTSS CTCCAGGTACTCCTGGCAGCGGTACTGCATC TGSPTTCCTCTCCAGGTGCTTCTCCGGGCACCAGCT CTACTGGTTCTCCA LCW0404_053_GSSTPSGATGSPGSSP 527 GGTAGCTCTACTCCTTCTGGTGCAACTGGTTC 571 GFP-N_D11.ab1SASTGTGPGASPGTSS TCCAGGTTCTAGCCCGTCTGCATCCACTGGT TGSPACCGGTCCAGGTGCTTCCCCTGGCACCAGCT CTACCGGTTCTCCA LCW0404_057_GASPGTSSTGSPGSST 528 GGTGCATCTCCTGGTACTAGCTCTACTGGTTC 572 GFP-N_E11.ab1PSGATGSPGSSPSAST TCCAGGTAGCTCTACTCCGTCTGGTGCAACC GTGPGGCTCTCCAGGTTCTAGCCCTTCTGCATCTAC CGGTACTGGTCCA LCW0404_060_GTPGSGTASSSPGSST 529 GGTACTCCTGGCAGCGGTACCGCATCTTCCT 573 GFP-N_F11.ab1PSGATGSPGASPGTSS CTCCAGGTAGCTCTACTCCGTCTGGTGCAAC TGSPTGGTTCCCCAGGTGCTTCTCCGGGTACCAGC TCTACCGGTTCTCCA LCW0404_062_GSSTPSGATGSPGTPG 530 GGTAGCTCTACCCCGTCTGGTGCAACCGGCT 574 GFP-N_G11.ab1SGTASSSPGSSTPSGA CCCCAGGTACTCCTGGTAGCGGTACCGCTTC TGSPTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTG CTACCGGCTCCCCA LCW0404_066_GSSPSASTGTGPGSSP 531 GGTTCTAGCCCTTCTGCATCCACCGGTACCG 575 GFP-N_H11.ab1SASTGTGPGASPGTSS GCCCAGGTTCTAGCCCGTCTGCTTCTACCGG TGSPTACTGGTCCAGGTGCTTCTCCGGGTACTAGC TCTACTGGTTCTCCA LCW0404_067_GTPGSGTASSSPGSST 532 GGTACCCCGGGTAGCGGTACCGCTTCTTCTT 576 GFP-N_A12.ab1PSGATGSPGSNPSAST CTCCAGGTAGCTCTACTCCGTCTGGTGCTAC GTGPCGGCTCTCCAGGTTCTAACCCTTCTGCATCCA CCGGTACCGGCCCA LCW0404_068_GSSPSASTGTGPGSST 533 GGTTCTAGCCCTTCTGCATCTACTGGTACTGG 577 GFP-N_B12.ab1PSGATGSPGASPGTSS CCCAGGTAGCTCTACTCCTTCTGGTGCTACC TGSPGGCTCTCCAGGTGCTTCTCCGGGTACTAGCT CTACCGGTTCTCCA LCW0404_069_GSSTPSGATGSPGASP 534 GGTAGCTCTACCCCTTCTGGTGCAACCGGCT 578 GFP-N_C12.ab1GTSSTGSPGTPGSGTA CTCCAGGTGCATCCCCGGGTACCAGCTCTAC SSSPCGGTTCTCCAGGTACTCCGGGTAGCGGTACC GCTTCTTCCTCTCCA LCW0404_070_GSSTPSGATGSPGSST 535 GGTAGCTCTACTCCGTCTGGTGCAACCGGTT 579 GFP-N_D12.ab1PSGATGSPGSSTPSGA CCCCAGGTAGCTCTACCCCTTCTGGTGCAAC TGSPCGGCTCCCCAGGTAGCTCTACCCCTTCTGGT GCAACTGGCTCTCCA LCW0404_073_GASPGTSSTGSPGTPG 536 GGTGCTTCTCCTGGCACTAGCTCTACCGGTTC 580 GFP-N_E12.ab1SGTASSSPGSSTPSGA TCCAGGTACCCCTGGTAGCGGTACCGCATCT TGSPTCCTCTCCAGGTAGCTCTACTCCTTCTGGTGC TACTGGTTCCCCA LCW0404_075_GSSTPSGATGSPGSSP 537 GGTAGCTCTACCCCGTCTGGTGCTACTGGCT 581 GFP-N_F12.ab1SASTGTGPGSSPSAST CCCCAGGTTCTAGCCCTTCTGCATCCACCGG GTGPTACCGGTCCAGGTTCTAGCCCGTCTGCATCT ACTGGTACTGGTCCA LCW0404_080_GASPGTSSTGSPGSSP 538 GGTGCTTCCCCGGGCACCAGCTCTACTGGTT 582 GFP-N_G12.ab1SASTGTGPGSSPSAST CTCCAGGTTCTAGCCCGTCTGCTTCTACTGGT GTGPACTGGTCCAGGTTCTAGCCCTTCTGCTTCCAC TGGTACTGGTCCA LCW0404_081_GASPGTSSTGSPGSSP 539 GGTGCTTCCCCGGGTACCAGCTCTACCGGTT 583 GFP-N_H12.ab1SASTGTGPGTPGSGT CTCCAGGTTCTAGCCCTTCTGCTTCTACCGGT ASSSPACCGGTCCAGGTACCCCTGGCAGCGGTACCG CATCTTCCTCTCCA

Example 5: Construction of XTEN_AE864

XTEN_AE864 was constructed from serial dimerization of XTEN_AE36 toAE72, 144, 288, 576 and 864. A collection of XTEN_AE72 segments wasconstructed from 37 different segments of XTEN_AE36. Cultures of E. coliharboring all 37 different 36-amino acid segments were mixed and plasmidwas isolated. This plasmid pool was digested with BsaI/NcoI to generatethe small fragment as the insert. The same plasmid pool was digestedwith BbsI/NcoI to generate the large fragment as the vector. The insertand vector fragments were ligated resulting in a doubling of the lengthand the ligation mixture was transformed into BL21Gold(DE3) cells toobtain colonies of XTEN_AE72.

This library of XTEN_AE72 segments was designated LCW0406. All clonesfrom LCW0406 were combined and dimerized again using the same process asdescribed above yielding library LCW0410 of XTEN_AE144. All clones fromLCW0410 were combined and dimerized again using the same process asdescribed above yielding library LCW0414 of XTEN_AE288. Two isolatesLCW0414.001 and LCW0414.002 were randomly picked from the library andsequenced to verify the identities. All clones from LCW0414 werecombined and dimerized again using the same process as described aboveyielding library LCW0418 of XTEN_AE576. We screened 96 isolates fromlibrary LCW0418 for high level of GFP fluorescence. 8 isolates withright sizes of inserts by PCR and strong fluorescence were sequenced and2 isolates (LCW0418.018 and LCW0418.052) were chosen for future usebased on sequencing and expression data.

The specific clone pCW0432 of XTEN_AE864 was constructed by combiningLCW0418.018 of XTEN_AE576 and LCW0414.002 of XTEN_AE288 using the samedimerization process as described above.

Example 6: Construction of XTEN_AM144

A collection of XTEN_AM144 segments was constructed starting from 37different segments of XTEN_AE36, 44 segments of XTEN_AF36, and 44segments of XTEN_AG36.

Cultures of E. coli that harboring all 125 different 36-amino acidsegments were mixed and plasmid was isolated. This plasmid pool wasdigested with BsaI/NcoI to generate the small fragment as the insert.The same plasmid pool was digested with BbsI/NcoI to generate the largefragment as the vector. The insert and vector fragments were ligatedresulting in a doubling of the length and the ligation mixture wastransformed into BL21Gold(DE3) cells to obtain colonies of XTEN_AM72.

This library of XTEN_AM72 segments was designated LCW0461. All clonesfrom LCW0461 were combined and dimerized again using the same process asdescribed above yielding library LCW0462. 1512 Isolates from libraryLCW0462 were screened for protein expression. Individual colonies weretransferred into 96 well plates and cultured overnight as startercultures. These starter cultures were diluted into fresh autoinductionmedium and cultured for 20-30 h. Expression was measured using afluorescence plate reader with excitation at 395 nm and emission at 510nm. 192 isolates showed high level expression and were submitted to DNAsequencing. Most clones in library LCW0462 showed good expression andsimilar physicochemical properties suggesting that most combinations ofXTEN_AM36 segments yield useful XTEN sequences. 30 isolates from LCW0462were chosen as a preferred collection of XTEN_AM144 segments for theconstruction of multifunctional proteins that contain multiple XTENsegments. The file names of the nucleotide and amino acid constructs andthe sequences for these segments are listed in Table 15.

TABLE 15 DNA and amino acid sequences for AM144 segments SEQ SEQ IDProtein ID Clone Sequence Trimmed NO: Sequence NO: LCW462_r1GGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAG 584 GTPGSGTASSSPG 617GTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGG SSTPSGATGSPGSTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGGT STPSGATGSPGSPAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTA AGSPTSTLEGTSECTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTAC SATPESGPGTSTECTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTTCT PSEGSAPGSSPSAAGCCCTTCTGCATCCACCGGTACCGGCCCAGGTTCTA STGTGPGSSPSASGCCCGTCTGCTTCTACCGGTACTGGTCCAGGTGCTTCT TGTGPGASPGTSSCCGGGTACTAGCTCTACTGGTTCTCCAGGTACCTCTA TGSPGTSTEPSEGCCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTAC SAPGTSTEPSEGSTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACC APGSEPATSGSETGGCAACCTCCGGTTCTGAAACTCCA P LCW462_r5GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCAG 585 GSTSESPSGTAPG 618GTTCTACTAGCGAATCCCCTTCTGGTACCGCACCAGG STSESPSGTAPGTTACTTCTCCGAGCGGCGAATCTTCTACTGCTCCAGGT SPSGESSTAPGTSACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTA TEPSEGSAPGTSTCCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTAC EPSEGSAPGTSESTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTGCA ATPESGPGASPGTTCTCCTGGTACCAGCTCTACCGGTTCTCCAGGTAGCTC SSTGSPGSSTPSGTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCC ATGSPGASPGTSSCGGGTACCAGCTCTACCGGTTCTCCAGGTTCTACTAG TGSPGSTSESPSGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGC TAPGSTSESPSGTGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCC APGTSTPESGSASCTGAAAGCGGTTCCGCTTCTCCA P LCW462_r9GGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAG 586 GTSTEPSEGSAPG 619GTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGG TSESATPESGPGTTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGT SESATPESGPGTSACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA TEPSEGSAPGTSECTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTAC SATPESGPGTSTETTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACT PSEGSAPGTSTEPTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCG SEGSAPGSEPATSAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCC GSETPGSPAGSPTGGCTGGCTCTCCGACCTCCACCGAGGAAGGTGCTTCT STEEGASPGTSSTCCTGGCACCAGCTCTACTGGTTCTCCAGGTTCTAGCC GSPGSSPSASTGTCTTCTGCTTCTACCGGTACTGGTCCAGGTTCTAGCCCT GPGSSPSASTGTGTCTGCATCCACTGGTACTGGTCCA P LCW462_r10GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAG 587 GSEPATSGSETPG 620GTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCAGG TSESATPESGPGTTACTTCTGAAAGCGCTACTCCGGAATCCGGTCCAGGT SESATPESGPGSTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTT SESPSGTAPGSTSCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTAC ESPSGTAPGTSPSTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTGCA GESSTAPGASPGTTCTCCGGGTACTAGCTCTACCGGTTCTCCAGGTTCTAG SSTGSPGSSPSASCCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTCT TGTGPGSSTPSGAACCCCGTCTGGTGCTACTGGTTCCCCAGGTAGCTCTA TGSPGSSTPSGATCTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTAC GSPGSSTPSGATGTCCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTG SPGASPGTSSTGSGCACCAGCTCTACCGGTTCTCCA P LCW462_r15GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAG 588 GASPGTSSTGSPG 621GTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGG SSPSASTGTGPGSTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGT STPSGATGSPGTSACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTA ESATPESGPGSEPGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAG ATSGSETPGSEPACGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTACT TSGSETPGTSESATCTGAAAGCGCTACTCCGGAGTCCGGTCCAGGTACCT TPESGPGTSTEPSCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACTTC EGSAPGTSTEPSETACTGAACCTTCTGAGGGTAGCGCTCCAGGTACCTCT GSAPGTSTEPSEGACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTA SAPGTSTEPSEGSCTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACC APGSEPATSGSETGGCAACCTCCGGTTCTGAAACTCCA P LCW462_r16GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAG 589 GTSTEPSEGSAPG 622GTAGCCCGGCAGGTTCTCCTACTTCCACTGAGGAAGG SPAGSPTSTEEGTTACTTCTACCGAACCTTCTGAGGGTAGCGCACCAGGT STEPSEGSAPGTSACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTA ESATPESGPGSEPGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTAC ATSGSETPGTSESCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGC ATPESGPGSPAGSCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTT PTSTEEGTSESATCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTC PESGPGTSTEPSETACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCGA GSAPGSEPATSGSACCTGCTACTTCTGGTTCTGAAACTCCAGGTACTTCTA ETPGTSTEPSEGSCCGAACCGTCCGAGGGTAGCGCTCCAGGTAGCGAAC APGSEPATSGSETCTGCTACTTCTGGTTCTGAAACTCCA P LCW462_r20GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAG 590 GTSTEPSEGSAPG 623GTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGG TSTEPSEGSAPGTTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGT STEPSEGSAPGTSACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTA TEPSEGSAPGTSTCCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTAC EPSEGSAPGTSTECTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACT PSEGSAPGTSTEPTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTT SEGSAPGTSESATCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTC PESGPGTSESATPTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCT ESGPGTSTEPSEGACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAAC SAPGSEPATSGSECTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGC TPGSPAGSPTSTETGGCTCTCCGACCTCCACCGAGGAA E LCW462_r23GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAG 591 GTSTEPSEGSAPG 624GTACTTCTACTGAACCTTCTGAAGGCAGCGCTCCAGG TSTEPSEGSAPGTTACTTCTACTGAACCTTCCGAAGGTAGCGCACCAGGT STEPSEGSAPGSTTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTTC SESPSGTAPGSTSTACCAGCGAATCCCCTTCTGGCACCGCACCAGGTACT ESPSGTAPGTSTPTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTAGCG ESGSASPGSEPATAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTC SGSETPGTSESATTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCT PESGPGTSTEPSEACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTA GSAPGTSTEPSEGCTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGA SAPGTSESATPESAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGA GPGTSESATPESGAAGCGCAACCCCGGAGTCCGGCCCA P LCW462_r24GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCTCCAG 592 GSSTPSGATGSPG 625GTTCTAGCCCGTCTGCTTCTACCGGTACCGGTCCAGG SSPSASTGTGPGSTAGCTCTACCCCTTCTGGTGCTACTGGTTCTCCAGGTA STPSGATGSPGSPGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAG AGSPTSTEEGSPACCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACT GSPTSTEEGTSTETCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTGCTT PSEGSAPGASPGTCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTTCTAG SSTGSPGSSPSASCCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCCG TGTGPGTPGSGTGGCAGCGGTACTGCTTCTTCCTCTCCAGGTTCTACTAG ASSSPGSTSSTAECTCTACTGCTGAATCTCCTGGCCCAGGTACTTCTCCTA SPGPGTSPSGESSGCGGTGAATCTTCTACCGCTCCAGGTACCTCTACTCC TAPGTSTPESGSAGGAAAGCGGTTCTGCATCTCCA SP LCW462_r27GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAG 593 GTSTEPSEGSAPG 626GTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGG TSESATPESGPGTTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGT STEPSEGSAPGTSACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTA TEPSEGSAPGTSECTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTAC SATPESGPGTSESCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACT ATPESGPGTPGSGCCTGGCAGCGGTACCGCTTCTTCTTCTCCAGGTGCTTC TASSSPGASPGTSTCCTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTCTC STGSPGASPGTSSCGGGCACTAGCTCTACTGGTTCTCCAGGTAGCCCTGC TGSPGSPAGSPTSTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCT TEEGSPAGSPTSTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCG EEGTSTEPSEGSAAACCTTCCGAAGGTAGCGCTCCA P LCW462_r28GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAG 594 GSPAGSPTSTEEG 627GTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGG TSTEPSEGSAPGTTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGT STEPSEGSAPGTSACCTCTACCGAACCGTCTGAAGGTAGCGCACCAGGTA TEPSEGSAPGTSECCTCTGAAAGCGCAACTCCTGAGTCCGGTCCAGGTAC SATPESGPGTSESTTCTGAAAGCGCAACCCCGGAGTCTGGCCCAGGTACC ATPESGPGTPGSGCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCT TASSSPGSSTPSGCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCT ATGSPGASPGTSSCCGGGCACCAGCTCTACCGGTTCTCCAGGTACCTCTA TGSPGTSTEPSEGCTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGA SAPGTSESATPESAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACT GPGTSTEPSEGSAGAACCGTCCGAAGGTAGCGCACCA P LCW462_r38GGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAG 595 GSEPATSGSETPG 628GTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCAGG TSESATPESGPGSTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCAGGT EPATSGSETPGSSAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTA TPSGATGSPGTPGCTCCTGGTAGCGGTACCGCTTCTTCTTCTCCAGGTAGC SGTASSSPGSSTPTCTACTCCGTCTGGTGCTACCGGCTCCCCAGGTGCAT SGATGSPGASPGTCTCCTGGTACCAGCTCTACCGGTTCTCCAGGTAGCTCT SSTGSPGSSTPSGACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCCC ATGSPGASPGTSSGGGTACCAGCTCTACCGGTTCTCCAGGTAGCGAACCT TGSPGSEPATSGSGCTACTTCTGGTTCTGAAACTCCAGGTACTTCTACCG ETPGTSTEPSEGSAACCGTCCGAGGGTAGCGCTCCAGGTAGCGAACCTG APGSEPATSGSETCTACTTCTGGTTCTGAAACTCCA P LCW462_r39GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAG 596 GTSTEPSEGSAPG 629GTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAG TSTEPSEGSAPGTGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGG SESATPESGPGSPTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGT AGSPTSTEEGSPAAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTA GSPTSTEEGTSTECTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTAG PSEGSAPGSPAGSCCCGGCTGGTTCTCCGACTTCCACCGAGGAAGGTACC PTSTEEGTSTEPSTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTACCT EGSAPGTSTEPSECTACTGAACCTTCCGAAGGCAGCGCTCCAGGTGCTTC GSAPGASPGTSSTCCCGGGCACCAGCTCTACTGGTTCTCCAGGTTCTAGC GSPGSSPSASTGTCCGTCTGCTTCTACTGGTACTGGTCCAGGTTCTAGCCC GPGSSPSASTGTGTTCTGCTTCCACTGGTACTGGTCCA P LCW462_r41GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAG 597 GSSTPSGATGSPG 630GTGCTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGGT ASPGTSSTGSPGSAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTA STPSGATGSPGSPGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTAC AGSPTSTEEGTSECTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGC SATPESGPGSEPAGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTGCAT TSGSETPGASPGTCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTAGCTCT SSTGSPGSSTPSGACTCCGTCTGGTGCAACCGGCTCTCCAGGTTCTAGCC ATGSPGSSPSASTCTTCTGCATCTACCGGTACTGGTCCAGGTTCTACCAG GTGPGSTSESPSGCGAATCCCCTTCTGGTACTGCTCCAGGTTCTACCAGC TAPGSTSESPSGTGAATCCCCTTCTGGCACCGCACCAGGTACTTCTACCC APGTSTPESGSASCTGAAAGCGGCTCCGCTTCTCCA P LCW462_r42GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAG 598 GSTSESPSGTAPG 631GTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGG STSESPSGTAPGTTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGT SPSGESSTAPGTSACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTA ESATPESGPGTSTCCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAC EPSEGSAPGTSTETTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACC PSEGSAPGTSTEPTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTT SEGSAPGTSESATCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTC PESGPGTSTEPSETACTGAACCGTCCGAAGGTAGCGCACCAGGTAGCTCT GSAPGSSTPSGATACCCCGTCTGGTGCTACCGGTTCCCCAGGTGCTTCTCC GSPGASPGTSSTGTGGTACTAGCTCTACCGGTTCTCCAGGTAGCTCTACC SPGSSTPSGATGSCCGTCTGGTGCTACTGGCTCTCCA P LCW462_r43GGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCAG 599 GSTSSTAESPGPG 632GTACCTCTCCTAGCGGTGAATCTTCTACCGCTCCAGG TSPSGESSTAPGTTACTTCTCCGAGCGGTGAATCTTCTACCGCTCCAGGTT SPSGESSTAPGSTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAGGTTC SSTAESPGPGSTSTACCAGCTCTACTGCAGAATCTCCTGGCCCAGGTACT STAESPGPGTSTPTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTACTT ESGSASPGTSPSGCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTTCTACC ESSTAPGSTSSTAAGCTCTACTGCTGAATCTCCTGGCCCAGGTACTTCTA ESPGPGTSTPESGCCCCGGAAAGCGGCTCCGCTTCTCCAGGTTCTACCAG SASPGSTSSTAESCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACTAGC PGPGSTSESPSGTGAATCTCCGTCTGGCACCGCACCAGGTACTTCCCCTA APGTSPSGESSTAGCGGTGAATCTTCTACTGCACCA P LCW462_r45GGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCAG 600 GTSTPESGSASPG 633GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAGG STSESPSGTAPGSTTCTACTAGCTCTACTGCTGAATCTCCGGGCCCAGGT TSSTAESPGPGTSACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTA TEPSEGSAPGTSTCCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTAC EPSEGSAPGTSESTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTACC ATPESGPGTSESATCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCT TPESGPGTSTEPSCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTC EGSAPGTSTEPSETACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCT GSAPGTSESATPEGAAAGCGCTACTCCGGAGTCCGGTCCAGGTACCTCTA SGPGTSTEPSEGSCCGAACCGTCCGAAGGCAGCGCTCCAGGTACTTCTAC APGTSTEPSEGSATGAACCTTCTGAGGGTAGCGCTCCC P LCW462_r47GGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAG 601 GTSTEPSEGSAPG 634GTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGG TSTEPSEGSAPGSTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCAGGT EPATSGSETPGTSACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTA TEPSEGSAPGTSECTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTAC SATPESGPGTSESCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTGC ATPESGPGASPGTATCTCCGGGTACTAGCTCTACCGGTTCTCCAGGTTCTA SSTGSPGSSPSASGCCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTC TGTGPGSSTPSGATACCCCGTCTGGTGCTACTGGTTCCCCAGGTAGCTCT TGSPGSSTPSGATACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTA GSPGSSTPSGATGCTCCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCT SPGASPGTSSTGSGGCACCAGCTCTACCGGTTCTCCA P LCW462_r54GGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAG 602 GSEPATSGSETPG 635GTAGCGAACCTGCAACCTCCGGCTCTGAAACCCCAGG SEPATSGSETPGTTACTTCTACTGAACCTTCTGAGGGCAGCGCACCAGGT STEPSEGSAPGSEAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTA PATSGSETPGTSECCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTAC SATPESGPGTSTETTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGC PSEGSAPGSSTPSTCTACTCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTC GATGSPGSSTPSGTACCCCTTCTGGTGCAACCGGCTCCCCAGGTGCTTCTC ATGSPGASPGTSSCGGGTACCAGCTCTACTGGTTCTCCAGGTAGCTCTAC TGSPGSSTPSGATCCCGTCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTG GSPGASPGTSSTGGTACTAGCTCTACCGGTTCTCCAGGTAGCTCTACCCC SPGSSTPSGATGSGTCTGGTGCTACTGGCTCTCCA P LCW462_r55GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAG 603 GTSTEPSEGSAPG 636GTACTTCTACTGAACCTTCTGAAGGCAGCGCTCCAGG TSTEPSEGSAPGTTACTTCTACTGAACCTTCCGAAGGTAGCGCACCAGGT STEPSEGSAPGTSACTTCTGAAAGCGCTACTCCGGAGTCCGGTCCAGGTA ESATPESGPGTSTCCTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTAC EPSEGSAPGTSTETTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTTCT PSEGSAPGSTSESACTAGCGAATCTCCGTCTGGCACTGCTCCAGGTACTT PSGTAPGTSPSGECTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCC SSTAPGTSPSGESCCTAGCGGCGAATCTTCTACCGCTCCAGGTAGCCCGG STAPGSPAGSPTSCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGA TEEGTSESATPESAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACT GPGTSTEPSEGSAGAACCGTCCGAAGGTAGCGCTCCA P LCW462_r57GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAG 604 GTSTEPSEGSAPG 637GTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGG SEPATSGSETPGSTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGT PAGSPTSTEEGSPAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTA AGSPTSTEEGTSECTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAC SATPESGPGTSTECTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACC PSEGSAPGTSTEPTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCT SEGSAPGTSTEPSCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTC EGSAPGTSESATPTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCTCT ESGPGSSTPSGATACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCC GSPGSSPSASTGTCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCC GPGASPGTSSTGSGGGCACCAGCTCTACTGGTTCTCCA P LCW462_r61GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCAG 605 GSEPATSGSETPG 638GTAGCCCTGCTGGCTCTCCGACCTCTACCGAAGAAGG SPAGSPTSTEEGTTACCTCTGAAAGCGCTACCCCTGAGTCTGGCCCAGGT SESATPESGPGTSACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTA TEPSEGSAPGTSTCCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTAC EPSEGSAPGTSESTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTACC ATPESGPGTSTPETCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTTCTA SGSASPGSTSESPCCAGCGAATCCCCGTCTGGCACCGCACCAGGTTCTAC SGTAPGSTSSTAETAGCTCTACTGCTGAATCTCCGGGCCCAGGTACTTCT SPGPGTSESATPEGAAAGCGCTACTCCGGAGTCCGGTCCAGGTACCTCTA SGPGTSTEPSEGSCCGAACCGTCCGAAGGCAGCGCTCCAGGTACTTCTAC APGTSTEPSEGSATGAACCTTCTGAGGGTAGCGCTCCA P LCW462_r64GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAG 606 GTSTEPSEGSAPG 639GTACTTCTACTGAACCTTCTGAAGGCAGCGCTCCAGG TSTEPSEGSAPGTTACTTCTACTGAACCTTCCGAAGGTAGCGCACCAGGT STEPSEGSAPGTSACCTCTACCGAACCGTCTGAAGGTAGCGCACCAGGTA TEPSEGSAPGTSECCTCTGAAAGCGCAACTCCTGAGTCCGGTCCAGGTAC SATPESGPGTSESTTCTGAAAGCGCAACCCCGGAGTCTGGCCCAGGTACT ATPESGPGTPGSGCCTGGCAGCGGTACCGCATCTTCCTCTCCAGGTAGCT TASSSPGSSTPSGCTACTCCGTCTGGTGCAACTGGTTCCCCAGGTGCTTCT ATGSPGASPGTSSCCGGGTACCAGCTCTACCGGTTCTCCAGGTTCCACCA TGSPGSTSSTAESGCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCTCCT PGPGTSPSGESSTAGCGGTGAATCTTCTACTGCTCCAGGTACTTCTACTCC APGTSTPESGSASTGAAAGCGGCTCTGCTTCTCCA P LCW462_r67GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAG 607 GSPAGSPTSTEEG 640GTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGG TSESATPESGPGTTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGT STEPSEGSAPGTSACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTA ESATPESGPGSEPGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTAC ATSGSETPGTSTETTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTAGC PSEGSAPGSPAGSCCGGCTGGTTCTCCGACTTCCACCGAGGAAGGTACCT PTSTEEGTSTEPSCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTACCTC EGSAPGTSTEPSETACTGAACCTTCCGAAGGCAGCGCTCCAGGTACTTCT GSAPGTSTEPSEGACCGAACCGTCCGAGGGCAGCGCTCCAGGTACTTCTA SAPGTSTEPSEGSCTGAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTAC APGTSTEPSEGSATGAACCTTCCGAAGGTAGCGCACCA P LCW462_r69GGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAG 608 GTSPSGESSTAPG 641GTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAGG STSSTAESPGPGTTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGT SPSGESSTAPGTSACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTA ESATPESGPGTSTCCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAC EPSEGSAPGTSTETTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTTCT PSEGSAPGSSPSAAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTAGCT STGTGPGSSTPSGCTACTCCTTCTGGTGCTACCGGCTCTCCAGGTGCTTCT ATGSPGASPGTSSCCGGGTACTAGCTCTACCGGTTCTCCAGGTACTTCTA TGSPGTSTPESGSCTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCC ASPGTSPSGESSTTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTA APGTSPSGESSTAGCGGCGAATCTTCTACTGCTCCA P LCW462_r70GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAG 609 GTSESATPESGPG 642GTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGG TSTEPSEGSAPGTTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGT STEPSEGSAPGSPAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTA AGSPTSTEEGSPAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTAC GSPTSTEEGTSTETTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTTCT PSEGSAPGSSPSAAGCCCTTCTGCTTCCACCGGTACTGGCCCAGGTAGCT STGTGPGSSTPSGCTACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGCTCT ATGSPGSSTPSGAACTCCTTCTGGTGCAACTGGCTCTCCAGGTAGCGAAC TGSPGSEPATSGSCGGCAACTTCCGGCTCTGAAACCCCAGGTACTTCTGA ETPGTSESATPESAAGCGCTACTCCTGAGTCTGGCCCAGGTAGCGAACCT GPGSEPATSGSETGCTACCTCTGGCTCTGAAACCCCA P LCW462_r72GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAG 610 GTSTEPSEGSAPG 643GTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGG TSTEPSEGSAPGTTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGT STEPSEGSAPGSSAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTG TPSGATGSPGASPCTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGGTAGC GTSSTGSPGSSTPTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTACTTC SGATGSPGTSESATGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGA TPESGPGSEPATSACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCT GSETPGTSTEPSEACCGAACCGTCCGAAGGTAGCGCACCAGGTTCTACTA GSAPGSTSESPSGGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAG TAPGSTSESPSGTCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACC APGTSTPESGSASCCTGAAAGCGGTTCCGCTTCTCCA P LCW462_r73GGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAG 611 GTSTPESGSASPG 644GTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAGG STSSTAESPGPGSTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCAGGTT TSSTAESPGPGSSCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTAG PSASTGTGPGSSTCTCTACTCCTTCTGGTGCTACCGGCTCTCCAGGTGCTT PSGATGSPGASPGCTCCGGGTACTAGCTCTACCGGTTCTCCAGGTAGCGA TSSTGSPGSEPATACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCT SGSETPGTSESATGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGG PESGPGSPAGSPTCAGGTTCTCCGACTTCCACTGAGGAAGGTTCTACTAG STEEGSTSESPSGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGC TAPGSTSESPSGTGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCC APGTSTPESGSASCTGAAAGCGGTTCCGCTTCTCCC P LCW462_r78GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAG 612 GSPAGSPTSTEEG 645GTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGG TSESATPESGPGTTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGT STEPSEGSAPGSTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTT SESPSGTAPGSTSCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTAC ESPSGTAPGTSPSTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTACC GESSTAPGTSTEPTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTAGCC SEGSAPGSPAGSPCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTC TSTEEGTSTEPSETACCGAACCTTCTGAGGGTAGCGCACCAGGTAGCGA GSAPGSEPATSGSACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCT ETPGTSESATPESGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA GPGTSTEPSEGSACTGAACCGTCCGAGGGCAGCGCACCA P LCW462_r79GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAG 613 GTSTEPSEGSAPG 646GTAGCCCGGCAGGTTCTCCTACTTCCACTGAGGAAGG SPAGSPTSTEEGTTACTTCTACCGAACCTTCTGAGGGTAGCGCACCAGGT STEPSEGSAPGTSACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTA PSGESSTAPGTSPCCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTAC SGESSTAPGTSPSCTCCCCTAGCGGTGAATCTTCTACCGCACCAGGTTCT GESSTAPGSTSESACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTTCTA PSGTAPGSTSESPCCAGCGAATCCCCTTCTGGCACCGCACCAGGTACTTC SGTAPGTSTPESGTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTAGCGAA SASPGSEPATSGSCCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTG ETPGTSESATPESAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTAC GPGTSTEPSEGSATGAACCGTCCGAGGGCAGCGCACCA P LCW462_r87GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAG 614 GSEPATSGSETPG 647GTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCAGG TSESATPESGPGTTACTTCTGAAAGCGCTACTCCGGAATCCGGTCCAGGT SESATPESGPGTSACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTT PSGESSTAPGSTSCTACTAGCTCTACCGCTGAATCTCCGGGCCCAGGTAC STAESPGPGTSPSTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTTCTA GESSTAPGSTSESCTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTC PSGTAPGTSPSGECCCTAGCGGTGAATCTTCTACTGCTCCAGGTTCTACC SSTAPGSTSSTAEAGCTCTACCGCAGAATCTCCGGGTCCAGGTAGCTCTA SPGPGSSTPSGATCTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTAC GSPGSSTPSGATGCCCTTCTGGTGCAACCGGCTCCCCAGGTAGCTCTACC SPGSSTPSGANWCCTTCTGGTGCAAACTGGCTCTCC LS LCW462_r88GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAG 615 GSPAGSPTSTEEG 648GTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGG SPAGSPTSTEEGTTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGT STEPSEGSAPGTSACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTA TEPSEGSAPGTSTCCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTAC EPSEGSAPGTSESTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTGCA ATPESGPGASPGTTCTCCTGGTACCAGCTCTACCGGTTCTCCAGGTAGCTC SSTGSPGSSTPSGTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCC ATGSPGASPGTSSCGGGTACCAGCTCTACCGGTTCTCCAGGTAGCTCTAC TGSPGSSTPSGATCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGC GSPGTPGSGTASSAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCC SPGSSTPSGATGSTTCTGGTGCTACTGGCTCTCCA P LCW462_r89GGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAG 616 GSSTPSGATGSPG 649GTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGG TPGSGTASSSPGSTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTA STPSGATGSPGSPGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTAC AGSPTSTEEGTSETTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACC SATPESGPGTSTETCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTACCT PSEGSAPGTSESACTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGA TPESGPGSEPATSACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCT GSETPGTSESATPGAAAGCGCAACCCCGGAATCTGGTCCAGGTACTTCTA ESGPGTSTEPSEGCTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGA SAPGTSESATPESAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGA GPGTSESATPESGAAGCGCAACCCCGGAGTCCGGCCCA P

Example 7: Construction of XTEN_AM288

The entire library LCW0462 was dimerized as described in Example 6resulting in a library of XTEN_AM288 clones designated LCW0463. 1512isolates from library LCW0463 were screened using the protocol describedin Example 6. 176 highly expressing clones were sequenced and 40preferred XTEN_AM288 segments were chosen for the construction ofmultifunctional proteins that contain multiple XTEN segments with 288amino acid residues.

Example 8: Construction of XTEN_AM432

We generated a library of XTEN_AM432 segments by recombining segmentsfrom library LCW0462 of XTEN_AM144 segments and segments from libraryLCW0463 of XTEN_AM288 segments. This new library of XTEN_AM432 segmentwas designated LCW0464. Plasmids were isolated from cultures of E. coliharboring LCW0462 and LCW0463, respectively. 1512 isolates from libraryLCW0464 were screened using the protocol described in Example 6. 176highly expressing clones were sequenced and 39 preferred XTEN_AM432segment were chosen for the construction of longer XTENs and for theconstruction of multifunctional proteins that contain multiple XTENsegments with 432 amino acid residues.

In parallel we constructed library LMS0100 of XTEN_AM432 segments usingpreferred segments of XTEN_AM144 and XTEN_AM288. Screening this libraryyielded 4 isolates that were selected for further construction

Example 9: Construction of XTEN_AM875

The stuffer vector pCW0359 was digested with BsaI and KpnI to remove thestuffer segment and the resulting vector fragment was isolated byagarose gel purification.

We annealed the phosphorylated oligonucleotide BsaI-AscI-KpnI for P:AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO:650) and the non-phosphorylated oligonucleotide BsaI-AscI-KpnIrev:CCTCGAGTGAAGACGAACCTCCCGTGCTTGGCGCGCCGCTTGCGCTTGC (SEQ ID NO: 651) forintroducing the sequencing island A (SI-A) which encodes amino acidsGASASGAPSTG (SEQ ID NO: 652) and has the restriction enzyme AscIrecognition nucleotide sequence GGCGCGCC inside. The annealedoligonucleotide pairs were ligated with BsaI and KpnI digested stuffervector pCW0359 prepared above to yield pCW0466 containing SI-A. We thengenerated a library of XTEN_AM443 segments by recombining 43 preferredXTEN_AM432 segments from Example 8 and SI-A segments from pCW0466 atC-terminus using the same dimerization process described in Example 5.This new library of XTEN_AM443 segments was designated LCW0479.

We generated a library of XTEN_AM875 segments by recombining segmentsfrom library LCW0479 of XTEN_AM443 segments and 43 preferred XTEN_AM432segments from Example 8 using the same dimerization process described inexample 5. This new library of XTEN_AM875 segment was designatedLCW0481.

Example 10: Construction of XTEN_AM1318

We annealed the phosphorylated oligonucleotide BsaI-FseI-Kpnl for P:AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO:653) and the non-phosphorylated oligonucleotide BsaI-FseI-KpnIrev:CCTCGAGTGAAGACGAACCTCCGCTTGGGGCCGGCCCCGTTGGTTCTGG (SEQ ID NO: 654) forintroducing the sequencing island B (SI-B) which encodes amino acidsGPEPTGPAPSG (SEQ ID NO: 655) and has the restriction enzyme FseIrecognition nucleotide sequence GGCCGGCC inside. The annealedoligonucleotide pairs were ligated with BsaI and KpnI digested stuffervector pCW0359 as used in Example 9 to yield pCW0467 containing SI-B. Wethen generated a library of XTEN_AM443 segments by recombining 43preferred XTEN_AM432 segments from Example 8 and SI-B segments frompCW0467 at C-terminus using the same dimerization process described inexample 5. This new library of XTEN_AM443 segments was designatedLCW0480.

We generated a library of XTEN_AM1318 segments by recombining segmentsfrom library LCW0480 of XTEN_AM443 segments and segments from libraryLCW0481 of XTEN_AM875 segments using the same dimerization process as inexample 5. This new library of XTEN_AM1318 segment was designatedLCW0487.

Example 11: Construction of XTEN_AD864

Using the several consecutive rounds of dimerization, we assembled acollection of XTEN_AD864 sequences starting from segments of XTEN_AD36listed in Example 1. These sequences were assembled as described inExample 5. Several isolates from XTEN_AD864 were evaluated and found toshow good expression and excellent solubility under physiologicalconditions. One intermediate construct of XTEN_AD576 was sequenced. Thisclone was evaluated in a PK experiment in cynomolgus monkeys and ahalf-life of about 20 h was measured.

Example 12: Construction of XTEN_AF864

Using the several consecutive rounds of dimerization, we assembled acollection of XTEN_AF864 sequences starting from segments of XTEN_AF36listed in Example 3. These sequences were assembled as described inExample 5. Several isolates from XTEN_AF864 were evaluated and found toshow good expression and excellent solubility under physiologicalconditions. One intermediate construct of XTEN_AF540 was sequenced. Thisclone was evaluated in a PK experiment in cynomolgus monkeys and ahalf-life of about 20 h was measured. A full length clone of XTEN_AF864had excellent solubility and showed half-life exceeding 60 h incynomolgus monkeys. A second set of XTEN_AF sequences was assembledincluding a sequencing island as described in Example 9.

Example 13: Construction of XTEN_AG864

Using the several consecutive rounds of dimerization, we assembled acollection of XTEN_AG864 sequences starting from segments of XTEN_AD36listed in Example 1. These sequences were assembled as described inExample 5. Several isolates from XTEN_AG864 were evaluated and found toshow good expression and excellent solubility under physiologicalconditions. A full length clone of XTEN_AG864 had excellent solubilityand showed half-life exceeding 60 h in cynomolgus monkeys.

Example 14: Construction of N-Terminal Extensions of XTEN—Constructionand Screening of 12mer Addition Libraries

This example details a step in the optimization of the N-terminus of theXTEN protein to promote the initiation of translation to allow forexpression of XTEN fusions at the N-terminus of fusion proteins withoutthe presence of a helper domain. Historically expression of proteinswith XTEN at the N-terminus was poor, yielding values that wouldessentially undetectable in the GFP fluorescence assay (<25% of theexpression with the N-terminal CBD helper domain). To create diversityat the codon level, seven amino acid sequences were selected andprepared with a diversity of codons. Seven pairs of oligonucleotidesencoding 12 amino acids with codon diversities were designed, annealedand ligated into the NdeI/BsaI restriction enzyme digested stuffervector pCW0551 (Stuffer-XTEN_AM875-GFP), and transformed into E. coliBL21Gold(DE3) competent cells to obtain colonies of seven libraries. Theresulting clones have N-terminal XTEN 12mers fused in-frame toXTEN_AM875-GFP to allow use of GFP fluorescence for screening theexpression. Individual colonies from the seven created libraries werepicked and grown overnight to saturation in 500 μl of super broth mediain a 96 deep well plate. The number of colonies picked ranged fromapproximately half to a third of the theoretical diversity of thelibrary (see Table 16).

TABLE 16 Theoretical Diversity and Sampling Numbers for12mer Addition Libraries. The amino acid resi-dues with randomized codons are underlined. SEQ Theoreti- MotifAmino Acid ID cal Number Library Family Sequence NO: Diversity screenedLCW546 AE12 MASPAGSPTSTEE 656  572 2 plates  (168) LCW547 AE12MATSESATPESGP 657 1536 5 plates  (420) LCW548 AF12 MATSPSGESSTAP 658 192 2 plates  (168) LCW549 AF12 MESTSSTAESPGP 659  384 2 plates  (168)LCW552 AG12 MASSTPSGATGSP 660  384 2 plates  (168) LCW553 AG12MEASPGTSSTGSP 661  384 2 plates  (168) LCW554 (CBD- MASTPESGSSG 662   321 plate  like)  (84)

The saturated overnight cultures were used to inoculate fresh 500 μlcultures in auto-induction media in which they were grown overnight at26° C. These expression cultures were then assayed using a fluorescenceplate reader (excitation 395 nm, emission 510 nm) to determine theamount of GFP reporter present (see FIG. 11 for results of expressionassays). The results, graphed as box and whisker plots, indicate thatwhile median expression levels were approximately half of the expressionlevels compared to the “benchmark” CBD N-terminal helper domain, thebest clones from the libraries were much closer to the benchmarks,indicating that further optimization around those sequences waswarranted. This is in contrast to previous XTEN versions that were <25%of the expression levels of the CBD N-terminal benchmark. The resultsalso show that the libraries starting with amino acids MA had betterexpression levels than those beginning with ME. This was most apparentwhen looking at the best clones, which were closer to the benchmarks asthey mostly start with MA. Of the 176 clones within 33% of the CBD-AM875benchmark, 87% begin with MA, where as only 75% of the sequences in thelibraries beginning with MA, a clear over representation of the clonesbeginning with MA at the highest level of expression. 96 of the bestclones were sequenced to confirm identity and twelve sequences (seeTable 17), 4 from LCW546, 4 from LCW547 and 4 from LCW552 were selectedfor further optimization.

TABLE 17 Advanced 12mer DNA Sequences SEQ ID Clone DNA Sequence NO:LCW546_02 ATGGCTAGTCCGGCTGGCTCTCCGACCTCCACT 663 GAGGAAGGTACTTCTACTLCW546_06 ATGGCTAGTCCTGCTGGCTCTCCAACCTCCACT 664 GAGGAAGGTACTTCTACTLCW546_07 ATGGCTAGTCCAGCAGGCTCTCCTACCTCCACC 665 GAGGAAGGTACTTCTACTLCW546_09 ATGGCTAGTCCTGCTGGCTCTCCGACCTCTACT 666 GAGGAAGGTACTTCTACTLCW547_03 ATGGCTACATCCGAAAGCGCAACCCCTGAGTCC 667 GGTCCAGGTACTTCTACTLCW547_06 ATGGCTACATCCGAAAGCGCAACCCCTGAATCT 668 GGTCCAGGTACTTCTACTLCW547_10 ATGGCTACGTCTGAAAGCGCTACTCCGGAATCT 669 GGTCCAGGTACTTCTACTLCW547_17 ATGGCTACGTCCGAAAGCGCTACCCCTGAATCC 670 GGTCCAGGTACTTCTACTLCW552_03 ATGGCTAGTTCTACCCCGTCTGGTGCAACCGGT 671 TCCCCAGGTACTTCTACTLCW552_05 ATGGCTAGCTCCACTCCGTCTGGTGCTACCGGT 672 TCCCCAGGTACTTCTACTLCW552_10 ATGGCTAGCTCTACTCCGTCTGGTGCTACTGGT 673 TCCCCAGGTACTTCTACTLCW552_11 ATGGCTAGTTCTACCCCTTCTGGTGCTACTGGT 674 TCTCCAGGTACTTCTACT

Example 15: Construction of N-Terminal Extensions of XTEN—Constructionand Screening of Libraries Optimizing Codons 3 and 4

This example details a step in the optimization of the N-terminus of theXTEN protein to promote the initiation of translation to allow forexpression of XTEN fusions at the N-terminus of proteins without thepresence of a helper domain. With preferences for the first two codonsestablished (see Example supra), the third and fourth codons wererandomized to determine preferences. Three libraries, based upon bestclones from LCW546, LCW547 and LCW552, were designed with the third andfourth residues modified such that all combinations of allowable XTENcodons were present at these positions (see FIG. 12). In order toinclude all the allowable XTEN codons for each library, nine pairs ofoligonucleotides encoding 12 amino acids with codon diversities of thirdand fourth residues were designed, annealed and ligated into theNdeI/BsaI restriction enzyme digested stuffer vector pCW0551(Stuffer-XTEN_AM875-GFP), and transformed into E. coli BL21Gold(DE3)competent cells to obtain colonies of three libraries LCW0569-571. With24 XTEN codons the theoretical diversity of each library is 576 uniqueclones. A total of 504 individual colonies from the three createdlibraries were picked and grown overnight to saturation in 500 μl ofsuper broth media in a 96 deep well plate. This provided sufficientcoverage to understand relative library performance and sequencepreferences. The saturated overnight cultures were used to inoculate new500 μl cultures in auto-induction media in which were grown overnight at26° C. These expression cultures were then assayed using a fluorescenceplate reader (excitation 395 nm, emission 510 nm) to determine theamount of GFP reporter present. The top 75 clones from the screen weresequenced and retested for GFP reporter expression versus the benchmarksamples (see FIG. 13). 52 clones yielded usable sequencing data and wereused for subsequent analysis. The results were broken down by libraryand indicate that LCW546 was the superior library. The results arepresented in Table 18. Surprisingly, it was discovered that base-linedfluorescence readings for the best clones were ˜900 AU, whereas the CBDN-terminal benchmark was only ˜600 AU. This indicates that this libraryhad instituted an approximately 33% improvement over the best clonesfrom the previous library which were approximately equal in expressionto the CBD N-terminal benchmark (Example 14).

TABLE 18 Third and Fourth Codon Optimization Library Comparison LCW569LCW570 LCW571 N 21 15 16 Mean Fluores- 628 491 537 cence (AU) SD 173 71232 CV 28% 15% 43%

Further trends were seen in the data showing preferences for particularcodons at the third and fourth position. Within the LCW569 library theglutamate codon GAA at the third position and the threonine codon ACTwere associated with higher expression as seen in Table 19.

TABLE 19 Preferred Third and Fourth Codons in LCW569 3 = GAA Rest 4 =ACT Rest N 8 13 4 17 Mean Fluores- 749 554 744 601 cence (AU) SD 234 47197 162 CV 31% 9% 26% 27%

Additionally, the retest of the top 75 clones indicated that severalwere now superior to the benchmark clones.

Example 16: Construction of N-Terminal Extensions of XTEN—Constructionand Screening of Combinatorial 12mer and 36mer Libraries

This example details a step in the optimization of the N-terminus of theXTEN protein to promote the initiation of translation to allow forexpression of XTEN fusions at the N-terminus of proteins without thepresence of a helper domain. With preferences for the first two codonsestablished (see Example supra), the N-terminus was examined in abroader context by combining the 12 selected 12mer sequences (seeExample supra) at the very N-terminus followed by 125 previouslyconstructed 36mer segments (see example supra) in a combinatorialmanner. This created novel 48mers at the N-terminus of the XTEN proteinand enabled the assessment of the impact of longer-range interactions atthe N-terminus on expression of the longer sequences (FIG. 14). Similarto the dimerization procedures used to assemble 36mers (see Exampleinfra), the plasmids containing the 125 selected 36mer segments weredigested with restriction enzymes BbsI/NcoI and the appropriate fragmentwas gel-purified. The plasmid from clone AC94 (CBD-XTEN_AM875-GFP) wasalso digested with BsaI/NcoI and the appropriate fragments weregel-purified. These fragments were ligated together and transformed intoE. coli BL21Gold(DE3) competent cells to obtain colonies of the libraryLCW0579, which also served as the vector for further cloning 12 selected12mers at the very N-terminus. The plasmids of LCW0579 were digestedwith NdeI/EcoRI/BsaI and the appropriate fragments were gel-purified. 12pairs of oligonucleotides encoding 12 selected 12mer sequences weredesigned, annealed and ligated with the NdeI/EcoRI/BsaI digested LCW0579vector, and transformed into E. coli BL21Gold(DE3) competent cells toobtain colonies of the library LCW0580. With a theoretical diversity of1500 unique clones, a total of 1512 individual colonies from the createdlibrary were picked and grown overnight to saturation in 500 μl of superbroth media in a 96 deep well plate. This provided sufficient coverageto understand relative library performance and sequence preferences. Thesaturated overnight cultures were used to inoculate new 500 μl culturesin auto-induction media that were grown overnight at 26° C. Theseexpression cultures were then assayed using a fluorescence plate reader(excitation 395 nm, emission 510 nm) to determine the amount of GFPreporter present. The top 90 clones were sequenced and retested for GFPreporter expression. 83 clones yielded usable sequencing data and wereused for subsequent analysis. The sequencing data was used to determinethe lead 12mer that was present in each clone and the impact of each12mer on expression was assessed. Clones LCW546_06 and LCW546_09 stoodout as being the superior N-terminus (see Table 20).

TABLE 20 Relative Performance of Clones Starting with LCW546_06 andLCW459_09 LCW546_06 All Others LCW546_09 All Others N 11 72 9 74 MeanFluores- 1100 752 988 775 cence (AU) SD 275 154 179 202 CV 25% 20% 18%26%

The sequencing and retest also revealed several instances of independentreplicates of the same sequence in the data producing similar results,thus increasing confidence in the assay. Additionally, 10 clones with 6unique sequences were superior to the benchmark clone. They arepresented in Table 21. It was noted that these were the only occurrencesof these sequences and in no case did one of these sequences occur andfail to beat the bench-mark clone. These six sequences were advanced forfurther optimization.

TABLE 21 Combinatorial 12mer and 36mer Clones Superior toBenchmark Clone SEQ Clone ID 12mer 36mer Name First 60 codons NO: NameName LCW580_ ATGGCTAGTCCTGCTGGCTCTC 675 LCW546_ LCW0404_ 51CAACCTCCACTGAGGAAGGTGC 06 040 ATCCCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCC CGTCTGGTGCTACCGGCTCTCC AGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTACTT CTACTGAACCGTCTGAAGGCAG CGCA LCW580_ATGGCTAGTCCTGCTGGCTCTC 676 LCW546_ LCW0404_ 81 CAACCTCCACTGAGGAAGGTGC 06040 ATCCCCGGGCACCAGCTCTACC GGTTCTCCAGGTAGCTCTACCC CGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGT GCTACTGGCTCTCCAGGTACTT CTACTGAACCGTCTGAAGGCAGCGCA LCW580_ ATGGCTAGTCCTGCTGGCTCTC 677 LCW546_ LCW0402_ 38CAACCTCCACTGAGGAAGGTAC 06 041 TTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAG GTTCTCCTACCTCCACCGAGGA AGGTACTTCTACCGAACCGTCCAGGGGTAGCGCACCAGGTACTT CTACTGAACCGTCTGAAGGCAG CGCA LCW580_ATGGCTAGTCCTGCTGGCTCTC 678 LCW546_ LCW0402_ 63 CGACCTCTACTGAGGAAGGTAC 09020 TTCTACTGAACCGTCTGAAGGC AGCGCACCAGGTAGCGAACCGG CTACTTCCGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCA ACTTCTACTGAAGAAGGTACTT CTACTGAACCGTCTGAAGGCAGCGCA LCW580_ ATGGCTAGTCCTGCTGGCTCTC 679 LCW546_ LCW0404_ 06CAACCTCCACTGAGGAAGGTAC 06 031 CCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCC CTTCTGGTGCAACCGGCTCTCC AGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTACTT CTACTGAACCGTCTGAAGGCAG CGCA LCW580_ATGGCTAGTCCTGCTGGCTCTC 680 LCW546_ LCW0402_ 35 CGACCTCTACTGAGGAAGGTAC 09020 TTCTACTGAACCGTCTGAAGGC AGCGCACCAGGTAGCGAACCGG CTACTTCCGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCA ACTTCTACTGAAGAAGGTACTT CTACTGAACCGTCTGAAGGCAGCGCA LCW580_ ATGGCTAGTCCTGCTGGCTCTC 681 LCW546_ LCW0403_ 67CGACCTCTACTGAGGAAGGTAC 09 064 CTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTA GCGGCGAATCTTCTACCGCTCC AGGTACCTCCCCTAGCGGTGAATCTTCTACCGCACCAGGTACTT CTACTGAACCGTCTGAAGGCAG CGCA LCW580_ATGGCTAGTCCTGCTGGCTCTC 682 LCW546_ LCW0403_ 13 CGACCTCTACTGAGGAAGGTAC 09060 CTCTACTCCGGAAAGCGGTTCC GCATCTCCAGGTTCTACCAGCG AATCCCCGTCTGGCACCGCACCAGGTTCTACTAGCTCTACTGCT GAATCTCCGGGCCCAGGTACTT CTACTGAACCGTCTGAAGGCAGCGCA LCW580_ ATGGCTAGTCCTGCTGGCTCTC 683 LCW546_ LCW0403_ 88CGACCTCTACTGAGGAAGGTAC 09 064 CTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTA GCGGCGAATCTTCTACCGCTCC AGGTACCTCCCCTAGCGGTGAATCTTCTACCGCACCAGGTACTT CTACTGAACCGTCTGAAGGCAG CGCA LCW580_ATGGCTAGTCCTGCTGGCTCTC 684 LCW546_ LCW0403_ 11 CGACCTCTACTGAGGAAGGTAC 09060 CTCTACTCCGGAAAGCGGTTCC GCATCTCCAGGTTCTACCAGCG AATCCCCGTCTGGCACCGCACCAGGTTCTACTAGCTCTACTGCT GAATCTCCGGGCCCAGGTACTT CTACTGAACCGTCTGAAGGCAGCGCA

Example 17: Construction of N-Terminal Extensions of XTEN—Constructionand Screening of Combinatorial 12mer and 36mer Libraries for XTEN-AM875and XTEN-AE864

This example details a step in the optimization of the N-terminus of theXTEN protein to promote the initiation of translation to allow forexpression of XTEN fusions at the N-terminus of proteins without thepresence of a helper domain. With preferences for the first four codons(see Examples supra, and for the best pairing of N-terminal 12mers and36mers (see Example supra) established, a combinatorial approach wasundertaken to examine the union of these preferences. This created novel48mers at the N-terminus of the XTEN protein and enabled the testing ofthe confluence of previous conclusions. Additionally, the ability ofthese leader sequences to be a universal solution for all XTEN proteinswas assessed by placing the new 48mers in front of both XTEN-AE864 andXTEN-AM875. Instead of using all 125 clones of 36mer segment, theplasmids from 6 selected clones of 36mer segment with best GFPexpression in the combinatorial library were digested withNdeI/EcoRI/BsaI and the appropriate fragments were gel-purified. Theplasmids from clones AC94 (CBD-XTEN_AM875-GFP) and AC104(CBD-XTEN_AE864-GFP) were digested with digested with NdeI/EcoRI/BsaIand the appropriate fragments were gel-purified. These fragments wereligated together and transformed into E. coli BL21Gold(DE3) competentcells to obtain colonies of the libraries LCW0585 (-XTEN_AM875-GFP) andLCW0586 (-XTEN_AE864-GFP), which could also serve as the vectors forfurther cloning 8 selected 12mers at the very N-terminus. The plasmidsof LCW0585 and LCW0586 were digested with NdeI/EcoRI/BsaI and theappropriate fragments were gel-purified. 8 pairs of oligonucleotidesencoding 8 selected 12mer sequences with best GFP expression in theprevious (Generation 2) screening were designed, annealed and ligatedwith the NdeI/EcoRI/BsaI digested LCW0585 and LCW0586 vectors, andtransformed into E. coli BL21Gold(DE3) competent cells to obtaincolonies of the final libraries LCW0587 (XTEN_AM923-GFP) and LCW0588(XTEN_AE912-GFP). With a theoretical diversity of 48 unique clones, atotal of 252 individual colonies from the created libraries were pickedand grown overnight to saturation in 500 μl of super broth media in a 96deep well plate. This provided sufficient coverage to understandrelative library performance and sequence preferences. The saturatedovernight cultures were used to inoculate new 500 μl cultures inauto-induction media in which were grown overnight at 26° C. Theseexpression cultures were then assayed using a fluorescence plate reader(excitation 395 nm, emission 510 nm) to determine the amount of GFPreporter present. The top 36 clones were sequenced and retested for GFPreporter expression. 36 clones yielded usable sequencing data and these36 were used for the subsequent analysis. The sequencing data determinedthe 12mer, the third codon, the fourth codon and the 36mer present inthe clone and revealed that many of the clones were independentreplicates of the same sequence. Additionally, the retest results forthese clones are close in value, indicating the screening process wasrobust. Preferences for certain combinations at the N-terminus were seenand were consistently yielding higher fluorescence values approximately50% greater than the benchmark controls (see Tables 22 and 23). Thesedate support the conclusion that the inclusion of the sequences encodingthe optimized N-terminal XTEN into the fusion protein genes conferred amarked enhancement on the expression of the fusion proteins.

TABLE 22 Preferred N-terminal Combinations for XTEN-AM875 Number ofClone Name Replicates 12mer 36mer Mean SD CV CBD-AM875 NA NA NA 1715 41816% LCW587_08 7 LCW546_06_3 = GAA LCW404_40 2333 572 18% LCW587_17 5LCW546_09_3 = GAA LCW403_64 2172 293 10%

TABLE 23 Preferred N-terminal Combinations for XTEN-AE864 Number ofClone Name Replicates 12mer 36mer Mean SD CV AC82 NA NA NA 1979 679 24%LCW588_14 8 LCW546_06_opt3 LCW404_31 2801 240  6% LCW588_27 2LCW546_06_opt34 LCW404_40 2839 556 15%

Notably, the preferred combination of the N-terminal for the XTEN-AM875and the preferred combination for the XTEN-AE864 are not the same(Tables 22 and 23), indicating more complex interactions further than150 bases from the initiation site influence expression levels. Thesequences for the preferred nucleotide sequences are listed in Table 24and the preferred clones were analyzed by SDS-PAGE to independentlyconfirm expression (see FIG. 15). The complete sequences of XTEN_AM923and XTEN_AE912 were selected for further analysis.

TABLE 24 Preferred DNA Sequences for first 48 AminoAcid Residues of N-terminal XTEN-AM875 and XTEN-AE864 XTEN SEQ CloneModi- ID Name fied Nucleotide Sequence NO: LCW587_ AM875ATGGCTGAACCTGCTGGCTCTCCAACCTCCACT 685 08GAGGAAGGTGCATCCCCGGGCACCAGCTCTACC GGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGT GCTACTGGCTCTCCAGGTACTTCTACTGAACCGTCTGAAGGCAGCGCA LCW587_ AM875 ATGGCTGAACCTGCTGGCTCTCCGACCTCTACT 686 17GAGGAAGGTACCTCCCCTAGCGGCGAATCTTCT ACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAA TCTTCTACCGCACCAGGTACTTCTACTGAACCGTCTGAAGGCAGCGCA LCW588_ AE864 ATGGCTGAACCTGCTGGCTCTCCAACCTCCACT 687 14GAGGAAGGTACCCCGGGTAGCGGTACTGCTTCT TCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGC TCTACCGGTTCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAG LCW588_ AE864 ATGGCTGAAACTGCTGGCTCTCCAACCTCCACT 688 27GAGGAAGGTGCATCCCCGGGCACCAGCTCTACC GGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGT GCTACTGGCTCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAG

Example 18: Construction of CTLA4-XTEN Genes and Vectors

The CTLA4 dimer genes encoding CTLA4(1-120)-XTEN_AE42-CTLA4(3-120) andCTLA4(1-125)-XTEN_AE42-CTLA4(3-125) were designed and synthesized byGeneArt, which introduced NdeI and BbsI restriction sites that arecompatible with the NdeI and BsaI sites that flank CBD (CelluloseBinding Domain) in the CBD-XTEN destination vector. The CBD-XTEN plasmidis a pET30 derivative from Novagen in the format of CBD-XTEN_AE864,where CBD is the stuffer for cloning. Constructs were generated byreplacing CBD in CBD-XTEN vector with the CTLA4 dimers-encodingfragments. The CBD-XTEN plasmid features a T7 promoter upstream of theCBD sequence, and an XTEN_AE864 sequence fused in-frame downstream ofthe CBD sequence. The stuffer CBD was removed by restriction digestionusing NdeI and BsaI endonucleases. Restriction endonucleases NdeI andBbsI digested CTLA4 dimer DNA fragments were ligated into the NdeI andBsaI digested CBD-XTEN vector using T4 DNA ligase and electroporatedinto BL21-Gold(DE3) (Stratagene). Transformants were screened by DNAminiprep and the desired constructs were confirmed by DNA sequencing.The final plasmids yield the CTLA4 dimers with XTEN_AE42 linker fused toXTEN_AE864 genes under the control of a T7 promoter. The resulting DNAare amino acid sequences are listed below. SEQ ID NO: 182 and 184.

The linker XTEN_AE42 included in the CTLA4 dimers-XTEN_AE864 plasmidswas removed by restriction digestion using the flanking AscI and FseIendonucleases. On the other end, XTEN_AE158 sequence with the sameflanking AscI and FseI restriction sites constructed on another plasmidwas digested with AscI and FseI endonucleases and ligated into the AscIand FseI digested CTLA4 dimers-XTEN_AE864 plasmids above using T4 DNAligase and electroporated into BL21-Gold(DE3) (Stratagene).Transformants were screened by DNA miniprep and the desired constructswere confirmed by DNA sequencing. The final plasmids yield the CTLA4dimers with XTEN_AE158 linker fused to XTEN_AE864 genes under thecontrol of a T7 promoter. The resulting DNA and amino acid sequences arepresented in Table 25 below.

Example 19: Construction of aIL6R-XTEN Genes and Vectors

DNA ligase and electroporated into BL21-Gold(DE3) (Stratagene). Theplasmids yield the aIL6R sscFv-XTEN_AE864 genes under the control of theT7 promoter. The plasmid with one additional BsaI site introduced by PCRwas further digested by NdeI and BsaI, ligated with NdeI and BsaIdigested XTEN_AE48 fragment using T4 DNA ligase and electroporated intoBL21-Gold (DE3). The final plasmid yields the XTEN_AE48-aIL6RscFv-XTEN_AE864 gene under the control of a T7 promoter. All thetransformants were screened by DNA miniprep and the desired constructswere confirmed by DNA sequencing. The resulting DNA sequences andencoded final product are provided below. The genes encoding aIL6R scFvat C-terminus were amplified by PCR, which introduced BsaI/HindIII andBsaI/BbsI&HindIII restriction sites that are compatible with the BbsIand HindIII sites that flank GFP (Green Fluorescent Protein) in theXTEN-GFP destination vector. The XTEN-GFP plasmid is a pET30 derivativefrom Novagen in the format of XTEN_AE912-GFP, where GFP is the stufferfor cloning. Constructs were generated by replacing GFP in XTEN-GFPvector with the aIL6R scFv-encoding PCR fragments. The XTEN-GFP plasmidfeatures a T7 promoter upstream of the XTEN_AE912 sequence and a stufferGFP sequence fused in-frame downstream of the XTEN_AE912 sequence. Thestuffer GFP was removed by restriction digestion using BbsI and HindIIIendonucleases. Restriction endonucleases BsaI and HindIII digested aIL6RscFv PCR fragments were ligated into the BbsI and HindIII digestedXTEN-GFP vector using T4 DNA ligase and electroporated intoBL21-Gold(DE3) (Stratagene). The plasmids yield the XTEN_AE912-aIL6RscFv genes under the control of the T7 promoter. The plasmid with oneadditional BbsI site introduced by PCR was further digested by BbsI andHindIII, ligated with BsaI and HindIII digested XTEN_AE144 fragmentusing T4 DNA ligase and electroporated into BL21-Gold (DE3). The finalplasmid yields the XTEN_AE912-aIL6R scFv-XTEN_ AE144 gene under thecontrol of a T7 promoter. All the transformants were screened by DNAminiprep and the desired constructs were confirmed by DNA sequencing.The resulting DNA sequences and encoded amino acid sequences areprovided in Table 25 below.

Example 20: Construction Anti-CD40-XTEN and XTEN-Anti-CD40 Genes andVectors

Construction anti-CD40-XTEN

Two genes encoding anti-CD40 were designed by reverse-translationcombined with codon optimization. These genes encoding anti-CD40 weresynthesized by GeneArt (Regensburg, Germany), which introduced NdeI andBbsI restriction sites that are compatible with the NdeI and BsaI sitesthat flank the stuffer in the pCBD-XTEN_AE864 destination vector. ThepCBD-XTEN_AE864 plasmid is a pET30 derivative from Novagen. Constructswere generated by replacing the CBD sequence in pCBD-XTEN_AE864 with theanti-CD40-encoding fragments. The pCBD-XTEN_AE864 features a T7 promoterupstream of CBD followed by an XTEN sequence fused in-frame. Restrictiondigested anti-CD40 DNA fragments were ligated into the cleavedpCBD-XTEN_AE864 vector using T4 DNA ligase and electroporated intoBL21(DE3) Gold (Stratagene, La Jolla, Calif.). Transformants werescreened by DNA miniprep and the desired construct was confirmed by DNAsequencing. The final vectors yield the anti-CD40-XTEN_AE864 gene underthe control of a T7 promoter. The resulting constructs are: AC384,pBC0009; AC385, pBC0010. The resulting DNA sequences and encoded aminoacid sequences are provided in Table 25 below.

Construction of XTEN-Anti-CD40

Two genes encoding anti-CD40 were amplified by polymerase chain reaction(PCR) using primers anti-CD40forBsaI (anti-CD40_1 forBsaI:ATAAAGGGTCTCCAGGTGAAATTGTTCTGACCCAATCTCC (SEQ ID NO: 689); anti-CD40_2forBsaI: ATAAAGGGTCTCCAGGTGAAATTGTTCTGACTCAATCTCCA (SEQ ID NO: 690)) andanti-CD40revHindIII (anti-CD40_1revHindIII:AACTCGAAGCTTttaGCTAGACACAGTAACCAGAGT (SEQ ID NO: 691); anti-CD40_2revHindIII: AACTCGAAGCTTttaAGAGGATACGGTCACCAGAGT (SEQ ID NO: 692)),which introduced BsaI and HindIII restriction sites that are compatiblewith the BbsI and HindIII sites that flank the stuffer in the XTEN_AE912destination vector. The XTEN_AE912-GFP plasmid is a pET30 derivativefrom Novagen. Constructs were generated by replacing the GFP sequence inXTEN_AE912-GFP with the anti-CD40-encoding fragments. The XTEN_AE912-GFPfeatures a T7 promoter upstream of XTEN followed by a GFP sequence fusedin-frame. The GFP fragments were removed by restriction digestion usingBbsI and HindIII endonucleases. Restriction digested anti-CD40 DNAfragments were ligated into the cleaved XTEN_AE912-GFP vector using T4DNA ligase and electroporated into BL21(DE3) Gold (Stratagene, La Jolla,Calif.). Transformants were screened by DNA miniprep and the desiredconstruct was confirmed by DNA sequencing. The final vectors yield theXTEN_AE912-anti-CD40 gene under the control of a T7 promoter. Theresulting constructs are: AC386, pBC0011; AC387, pBC0012. The resultingDNA sequences and encoded amino acid sequences are provided below.

Example 21: Construction of Anti-Her2-XTEN and XTEN-Anti-Her2 Genes andVectors

Construction of Anti-Her2-XTEN

The gene encoding scFv anti-Her2 has the format VL-XTEN_Y30-VH, where VLis the light chain of anti-Her2 antibody fragment, VH is the heavy chainof the antibody fragment and XTEN_Y30 is the sequenceGSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 1) flanked by the restrictionsites AgeI and KpnI. The gene was synthesized and cloned into acommercial vector for use as the PCR template. The gene encoding scFvanti-Her2 was amplified by polymerase chain reaction (PCR) using primersanti-Her2forNdeI (agatatacatATGGAAGACATTCAGATGACCCAGAGC (SEQ ID NO:693)) and anti-Her2revBsaI (CCGGGCTACCTGGAGACCCGGAAACAGTTACCAGAGTACC(SEQ ID NO: 694)), which introduced NdeI and BsaI restriction sites thatare compatible with the NdeI and BsaI sites that flank the stuffer inthe pCBD-XTEN_AE864 destination vector. The pCBD-XTEN_AE864 plasmid is apET30 derivative from Novagen. Constructs were generated by replacingthe CBD sequence in pCBD-XTEN_AE864 with the anti-Her2-encodingfragments. The pCBD-XTEN_AE864 features a T7 promoter upstream of CBDfollowed by an XTEN sequence fused in-frame. Restriction digestedanti-Her2 DNA fragments were ligated into the cleaved pCBD-XTEN_AE864vector using T4 DNA ligase and electroporated into BL21(DE3) Gold(Stratagene, La Jolla, Calif.). Transformants were screened by DNAminiprep and the desired construct was confirmed by DNA sequencing. Thefinal vectors yield the anti-Her2-XTEN_AE864 gene under the control of aT7 promoter. The resulting constructs is are: pBC0007, Seq ID 140. Theresulting DNA sequences and encoded amino acid sequences are providedbelow.

Construction of XTEN-Anti-Her2

The gene encoding svFV anti-Her2 (described above) was amplified bypolymerase chain reaction (PCR) using primers anti-Her2forBbsI(GCACCAGGTTCGTCTTCACTCGACATTCAGATGACCCAGAGC (SEQ ID NO: 695)) andanti-Her2revHindIII (AACTCGAAGCTTTCAGGAAACAGTTACCAGAGTACCTTG (SEQ ID NO:696)), which introduced BbsI and HindIII restriction sites that arecompatible with the BbsI and HindIII sites that flank the stuffer in theXTEN_AE912 destination vector. The XTEN_AE912-GFP plasmid is a pET30derivative from Novagen. Constructs were generated by replacing the GFPsequence in XTEN_AE912-GFP with the anti-Her2-encoding fragment. TheXTEN_AE912-GFP features a T7 promoter upstream of XTEN followed by a GFPsequence fused in-frame. The GFP fragment was removed by restrictiondigestion using BbsI and HindIII endonucleases. Restriction digestedanti-Her2 DNA fragment was ligated into the cleaved XTEN_AE912-GFPvector using T4 DNA ligase and electroporated into BL21(DE3) Gold(Stratagene, La Jolla, Calif.). Transformants were screened by DNAminiprep and the desired construct was confirmed by DNA sequencing. Thefinal vectors yield the XTEN_AE912-anti-Her2 gene under the control of aT7 promoter. The resulting constructs is: pBC0008. The resulting DNAsequences and encoded final product are provided in Table 25 below.

Example 22: Construction of Anti-EGFR-XTEN Genes and Vectors

The gene encoding anti-EGFR was amplified by polymerase chain reaction(PCR) from a library, which introduced NdeI and BbsI restriction sitesthat are compatible with the NdeI and BsaI sites that flank the firstFLAG tag in the FLAG-Y50-FLAG-His6 (“His6” disclosed as SEQ ID NO: 218)destination vector. Constructs were generated by replacing the FLAGsequence in FLAG-Y50-FLAG-His6 (“His6” disclosed as SEQ ID NO: 218) withthe anti-EGFR-encoding fragments. The FLAG-Y50-FLAG-His6 (“His6”disclosed as SEQ ID NO: 218) features a T7 promoter upstream of FLAGfollowed by the Y50-FLAG-His6 (“His6” disclosed as SEQ ID NO: 218)sequence fused in-frame. Restriction digested anti-EGFR DNA fragmentswere ligated into the cleaved FLAG-Y50-FLAG-His6 (“His6” disclosed asSEQ ID NO: 218) vector using T4 DNA ligase and electroporated into XL1Blue. Transformants were screened by DNA miniprep and the desiredconstructs were confirmed by DNA sequencing. The final vectors yield theanti-EGFR-Y50-FLAG-His6 (“His6” disclosed as SEQ ID NO: 218) gene underthe control of a T7 promoter. The resulting construct is: pMS0120. Theresulting DNA sequences and encoded amino acid sequences are provided inTable 25 below.

Example 23: Construction Anti-CD3-XTEN Genes and Vectors

The gene encoding anti-CD3 was amplified by polymerase chain reaction(PCR) from a library, which introduced NdeI and BbsI restriction sitesthat are compatible with the NdeI and BsaI sites that flank the stufferin the stuffer-Y288-GFP-His8 (“His8” disclosed as SEQ ID NO: 697)destination vector. Constructs were generated by replacing the stuffersequence in the stuffer-Y288-GFP-His8 (“His8” disclosed as SEQ ID NO:697) with the anti-CD3-encoding fragments. Restriction digested anti-CD3DNA fragments were ligated into the stuffer-Y288-GFP-His8 (“His8”disclosed as SEQ ID NO: 697) vector using T4 DNA ligase andelectroporated into BL21(DE3) Gold (Stratagene, La Jolla, Calif.).Transformants were screened by DNA miniprep and the desired constructswere confirmed by DNA sequencing. The final vectors yield theanti-CD3-Y288-GFP-His gene under the control of a T7 promoter. Theresulting construct is: pMS0185. The resulting DNA sequences and encodedamino acid sequences are provided below.

Example 24: Construction of Genes and Vectors Comprising Multiple scFv

Construction of the Anti-Her2-Y288-Anti-CD3-HA-His6 (“His6” Disclosed asSEQ ID NO: 218) and Anti-Her2-Y288-Anti-EGFR-HA-His6 (“His6” Disclosedas SEQ ID NO: 218) Genes and Vectors

The genes encoding anti-CD3 and anti-EGFR were amplified by polymerasechain reaction (PCR), which introduced BbsI restriction sites on bothends of the DNA fragments. A polymerase chain reaction (PCR) wasperformed to introduce an HA-His6 (“His6” disclosed as SEQ ID NO: 218)tag with a HindIII restriction site on the 3′ end. The anti-CD3-HA-His6(“His6” disclosed as SEQ ID NO: 218) (or the anti-EGFR-HA-His6 (“His6”disclosed as SEQ ID NO: 218)) fragments were compatible with the BbsIand HindIII sites that flank the GFP-His8 in the anti-Her2-Y288-GFP-His8(“His8” disclosed as SEQ ID NO: 697) destination vector. Constructs weregenerated by replacing the GFP-His8 (“His8” disclosed as SEQ ID NO: 697)sequence in the anti-Her2-Y288-GFP-His8 (“His8” disclosed as SEQ ID NO:697) with the anti-CD3-HA-His6 (“His6” disclosed as SEQ ID NO: 218) oranti-EGFR-HA-His6-encoding fragments (“His6” disclosed as SEQ ID NO:218). Restriction digested anti-CD3-HA-His6 (“His6” disclosed as SEQ IDNO: 218) or anti-EGFR-HA-His6 (“His6” disclosed as SEQ ID NO: 218) DNAfragments were ligated into the anti-Her2-Y288-GFP-His8 (“His8”disclosed as SEQ ID NO: 697) vector using T4 DNA ligase andelectroporated into BL21(DE3) Gold (Stratagene, La Jolla, Calif.).Transformants were screened by DNA miniprep and the desired constructswere confirmed by DNA sequencing. The final vectors yield theanti-Her2-Y288-anti-CD3-HA-His6 (“His6” disclosed as SEQ ID NO: 218) andanti-Her2-Y288-anti-EGFR-HA-His6 (“His6” disclosed as SEQ ID NO: 218)gene under the control of a T7 promoter. The resulting constructs are:pMS0183, AC48 and pMS0184, AC49. The resulting DNA sequences and encodedfinal product are provided in Table 25 below.

Construction of the Anti-Her2-Y288-Anti-CD3-HA-His8 (“His8” Disclosed asSEQ ID NO: 697) Genes and Vectors

The gene encoding anti-CD3 was amplified by polymerase chain reaction(PCR), which introduced BbsI and Spel restriction sites that arecompatible with the BbsI and Spel sites that flank the GFP in theanti-Her2-Y288-GFP-HA-His8 (“His8” disclosed as SEQ ID NO: 697)destination vector. Constructs were generated by replacing the GFPsequence in the anti-Her2-Y288-GFP-HA-His8 (“His8” disclosed as SEQ IDNO: 697) with the anti-CD3-encoding fragments. Restriction digestedanti-CD3 or DNA fragments were ligated into theanti-Her2-Y288-GFP-HA-His8 (“His8” disclosed as SEQ ID NO: 697) vectorusing T4 DNA ligase and electroporated into XL1 Blue. Transformants werescreened by DNA miniprep and the desired constructs were confirmed byDNA sequencing. The final vectors yield theanti-Her2-Y288-anti-CD3-HA-His8 (“His8” disclosed as SEQ ID NO: 697)gene under the control of a T7 promoter. The resulting construct is:pMS0212, AC69. The resulting DNA sequences and encoded amino acidsequences are provided in Table 25 below.

Example 25: Construction of Multivalent aEGFR VHH Binders

A library LCW0501 of EGFR_VHH-XTEN_AM144 was constructed by using PCR onfour clones of previously codon-optimized library LMS109.005, 020, 038 &045 with amino acid and DNA sequences designatedNdeI_BsaI-EGFR_VHH1-XTEN_AM144-GFP6˜229-H8 (LMS109.005);NdeI_BsaI-EGFRVHH1-XTEN_AM144-GFP6˜229-H8 (LMS109.020);NdeI_BsaI-EGFR_VHH1-XTEN_AM144-GFP6˜229-H8) (LMS109.038); andNdeI_BsaI-EGFR_VHH1-XTEN_AM144-GFP6˜229-H8 (LMS109.045). The amino acidand nucleic acid sequences are provided below. LCW0501 has the genelibrary of EGFR_VHH-XTEN_AM144 with the flanking restriction sitesNdeI&BsaI and BbsI, fused to GFP-8×His-tag (“8×His” disclosed as SEQ IDNO: 697) on a vector of pET30 derivative from Novagen.

The plasmid of LCW0501 was digested with BsaI/HindIII to generate thesmall fragment as the insert and digested with BbsI/HindIII to generatethe large fragment as the vector. The insert and vector fragments wereligated and the ligation mixture was electroporated into BL21-Gold (DE3)cells to obtain transformants of LCW0502. LCW0502 is the gene library ofEGFR_VHH-XTEN_AM144 dimer with the same flanking restriction sites BsaIand BbsI fused to GFP-8×His-tag (“8×His” disclosed as SEQ ID NO: 697) onthe same vector.

The plasmid of LCW0502 was digested with BsaI/HindIII to generate thesmall fragment as the insert and digested with BbsI/HindIII to generatethe large fragment as the vector. The insert and vector fragments wereligated and the ligation mixture was electroporated into BL21-Gold (DE3)cells to obtain transformants of LCW0503. LCW0503 is the gene library ofEGFR_VHH-XTEN_AM144 tetramer with the same flanking restriction sitesBsaI and BbsI fused to GFP-8×His-tag (“8×His” disclosed as SEQ ID NO:697) on the same vector.

The plasmid of LCW0502 was digested with BsaI/HindIII to generate thesmall fragment as the insert and the plasmid of LCW0503 was digestedwith BbsI/HindIII to generate the large fragment as the vector. Theinsert and vector fragments were ligated and the ligation mixture waselectroporated into BL21-Gold (DE3) cells to obtain transformants ofLCW0504. LCW0504 is the gene library of EGFR_VHH-XTEN_AM144 hexamer withthe same flanking restriction sites BsaI and BbsI fused to GFP-8×His-tag(“8×His” disclosed as SEQ ID NO: 697) on the same vector.

The plasmid of LCW0503 was digested with BsaI/HindIII to generate thesmall fragment as the insert and digested with BbsI/HindIII to generatethe large fragment as the vector. The insert and vector fragments wereligated and the ligation mixture was electroporated into BL21-Gold (DE3)cells to obtain transformants of LCW0505. LCW0505 is the gene library ofEGFR_VHH-XTEN_AM144 octamer with the same flanking restriction sitesBsaI and BbsI fused to GFP-8×His-tag (“8×His” disclosed as SEQ ID NO:697) on the same vector.

The LCW501, LCW502, LCW503, LCW504 and LCW505 libraries were screened todetermine the best expression candidate for evaluation. The screen wasconducted as follows for all of the libraries. Colonies a transformationwere picked into 500 μl cultures of LB in 96 deep well plates and grownto saturation overnight. These cultures were stored at 4° C. after 40 μlof these cultures was used to inoculate 500 μl of auto-induction mediaand these cultures were grown at 26° C. for >24 hours. Following thegrowth the GFP fluorescence of 100 μl of these auto-induction mediacultures was measured using a fluorescence plate reader. The GFPfluorescence is proportional to protein expression and is therefore aread out of total expression. The highest expressing clones wereidentified, and a new 1 ml overnight was started in SB from the originalsaturated overnight of that clone. Mini-preps were performed with thesenew cultures to derived plasmids. The DNA and amino acid sequences areprovided in Table 25 below.

TABLE 25DNA and amino acid sequences of binding fusion protein constructs SEQSEQ Clone ID Amino Acid ID Name DNA Sequence NO: Sequence NO: CTLA4-ATGGCAATGCATGTTGCACAGCCTGCAGTTGTTCTGGC 698 MAMHVAQPAVV 723 AE36-AAGCAGCCGTGGTATTGCCAGCTTTGTTTGTGAATATGC LASSRGIASFVCE CTLA4-AAGTCCGGGTAAAGCAACCGAAGTTCGTGTTACCGTTC YASPGKATEVRV AE864,TGAGACAGGCAGATAGCCAGGTTACCGAAGTTTGTGCA TVLRQADSQVTE AC389GCAACCTATATGATGGGTAATGAACTGACCTTTCTGGA VCAATYMMGNETGATAGCATTTGTACCGGCACCAGCAGCGGTAATCAGG LTFLDDSICTGTSTTAATCTGACCATTCAGGGTCTGCGTGCAATGGATACC SGNQVNLTIQGLGGTCTGTATATTTGTAAAGTGGAACTGATGTATCCGCCT RAMDTGLYICKVCCGTATTATCTGGGTATTGGTAATGGCACCCAGATTTAT ELMYPPPYYLGIGGTTATTGATCCGGAAGGCGCGCCAGGTACAAGCGAAAG NGTQIYVIDPEGACGCAACACCGGAAAGCGGTCCGGGTAGCGAACCGGCA PGTSESATPESGPACCAGCGGTAGCGAAACACCGGGTACATCAACCGAAC GSEPATSGSETPGCGAGCGAAGGTAGCGCACCGGGGCCGGCCATGCATGT TSTEPSEGSAPGPGGCCCAGCCAGCCGTGGTGCTGGCAAGTTCACGCGGTA AMHVAQPAVVLTTGCATCATTTGTGTGCGAATATGCATCACCTGGTAAAG ASSRGIASFVCEYCCACAGAAGTGCGCGTAACAGTACTGCGTCAGGCCGAT ASPGKATEVRVTTCACAGGTGACAGAAGTTTGCGCTGCCACATACATGAT VLRQADSQVTEVGGGCAACGAGCTGACATTCCTGGACGATTCAATTTGTA CAATYMMGNELCTGGTACAAGCTCAGGCAATCAGGTGAACCTGACAATC TFLDDSICTGTSSCAAGGCCTGAGAGCTATGGACACAGGCCTGTACATCTG GNQVNLTIQGLRCAAAGTTGAGCTGATGTACCCTCCGCCTTATTACTTAGG AMDTGLYICKVECATTGGCAACGGTACACAGATCTATGTGATCGATCCTG LMYPPPYYLGIGAGGGAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAG NGTQIYVIDPEGGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCC SPAGSPTSTEEGTAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAG SESATPESGPGTSGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGT TEPSEGSAPGSPAACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTAC GSPTSTEEGTSTECTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTC PSEGSAPGTSTEPTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAAC SEGSAPGTSESATCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCG PESGPGSEPATSGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGG SETPGSEPATSGSCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCG ETPGSPAGSPTSTCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCG EEGTSESATPESGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTC PGTSTEPSEGSAPCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTA GTSTEPSEGSAPGCCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAG SPAGSPTSTEEGTGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGG STEPSEGSAPGTSCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGT TEPSEGSAPGTSECCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGC SATPESGPGTSTEGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGG PSEGSAPGTSESATCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTC TPESGPGSEPATSCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA GSETPGTSTEPSEGGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGG GSAPGTSTEPSEGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTA SAPGTSESATPESCCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGC GPGTSESATPESGCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTC PGSPAGSPTSTEETGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAAC GTSESATPESGPGCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTGAA SEPATSGSETPGTAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGA SESATPESGPGTSACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAAC TEPSEGSAPGTSTCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCG EPSEGSAPGTSTETCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTC PSEGSAPGTSTEPCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTG SEGSAPGTSTEPSAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAG EGSAPGTSTEPSEGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTC GSAPGSPAGSPTSCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTA TEEGTSTEPSEGSGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCT APGTSESATPESGGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGAC PGSEPATSGSETPTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTC GTSESATPESGPGCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCA SEPATSGSETPGTGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGG SESATPESGPGTSTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTA TEPSEGSAPGTSECTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGC SATPESGPGSPAGCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCC SPTSTEEGSPAGSGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGG PTSTEEGSPAGSPCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAA TSTEEGTSESATPAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGA ESGPGTSTEPSEGACCGTCTGAGGGCAGCGCACCAGGTACCTCTGAAAGCG SAPGTSESATPESCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACC GPGSEPATSGSETTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAAC PGTSESATPESGPCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTG GSEPATSGSETPGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT TSESATPESGPGTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGG STEPSEGSAPGSPCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCA AGSPTSTEEGTSECCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCC SATPESGPGSEPAGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAAC TSGSETPGTSESACCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCC TPESGPGSPAGSPCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAA TSTEEGSPAGSPTGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGG STEEGTSTEPSEGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTA SAPGTSESATPESCTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACT GPGTSESATPESGTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCT PGTSESATPESGPGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACC GSEPATSGSETPGGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGG SEPATSGSETPGSCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGC PAGSPTSTEEGTSTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCT TEPSEGSAPGTSTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTC EPSEGSAPGSEPATGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTG TSGSETPGTSESAGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT TPESGPGTSTEPSGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGG EGSAPG CAGCGCACCAGGTTAA CTLA4-ATGGCAATGCATGTTGCACAGCCTGCAGTTGTTCTGGC 699 MAMHVAQPAVV 724 AE36-AAGCAGCCGTGGTATTGCCAGCTTTGTTTGTGAATATGC LASSRGIASFVCE CTLA4-AAGTCCGGGTAAAGCAACCGAAGTTCGTGTTACCGTTC YASPGKATEVRV AE864,TGAGACAGGCAGATAGCCAGGTTACCGAAGTTTGTGCA TVLRQADSQVTE AC390GCAACCTATATGATGGGTAATGAACTGACCTTTCTGGA VCAATYMMGNETGATAGCATTTGTACCGGCACCAGCAGCGGTAATCAGG LTFLDDSICTGTSTTAATCTGACCATTCAGGGTCTGCGTGCAATGGATACC SGNQVNLTIQGLGGTCTGTATATTTGTAAAGTGGAACTGATGTATCCGCCT RAMDTGLYICKVCCGTATTATCTGGGTATTGGTAATGGCACCCAGATTTAT ELMYPPPYYLGIGGTTATTGATCCGGAACCGTGTCCGGATAGCGGCGCGCC NGTQIYVIDPEPCAGGTACAAGCGAAAGCGCAACACCGGAAAGCGGTCCG PDSGAPGTSESATGGTAGCGAACCGGCAACCAGCGGTAGCGAAACACCGG PESGPGSEPATSGGTACATCAACCGAACCGAGCGAAGGTAGCGCACCGGG SETPGTSTEPSEGGCCGGCCATGCATGTGGCCCAGCCAGCCGTGGTGCTGG SAPGPAMHVAQPCAAGTTCACGCGGTATTGCATCATTTGTGTGCGAATATG AVVLASSRGIASFCATCACCTGGTAAAGCCACAGAAGTGCGCGTAACAGTA VCEYASPGKATECTGCGTCAGGCCGATTCACAGGTGACAGAAGTTTGCGC VRVTVLRQADSQTGCCACATACATGATGGGCAACGAGCTGACATTCCTGG VTEVCAATYMMACGATTCAATTTGTACTGGTACAAGCTCAGGCAATCAG GNELTFLDDSICTGTGAACCTGACAATCCAAGGCCTGAGAGCTATGGACAC GTSSGNQVNLTIQAGGCCTGTACATCTGCAAAGTTGAGCTGATGTACCCTC GLRAMDTGLYICCGCCTTATTACTTAGGCATTGGCAACGGTACACAGATC KVELMYPPPYYLTATGTGATCGATCCTGAACCTTGCCCTGATTCAGGAGGT GIGNGTQIYVIDPAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTAC EPCPDSGGSPAGSTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTC PTSTEEGTSESATTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAG PESGPGTSTEPSECAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACT GSAPGSPAGSPTSGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGA TEEGTSTEPSEGSACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCG APGTSTEPSEGSACTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACT PGTSESATPESGPTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTC GSEPATSGSETPGCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGA SEPATSGSETPGSCCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCG PAGSPTSTEEGTSGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGG ESATPESGPGTSTCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTA EPSEGSAPGTSTEGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACC PSEGSAPGSPAGSGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGC PTSTEEGTSTEPSACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTC EGSAPGTSTEPSECAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCA GSAPGTSESATPEGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGG SGPGTSTEPSEGSTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTA APGTSESATPESGGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACT PGSEPATSGSETPTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTC GTSTEPSEGSAPGTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTG TSTEPSEGSAPGTAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGAA SESATPESGPGTSAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGG ESATPESGPGSPACTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCG GSPTSTEEGTSESCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACC ATPESGPGSEPATTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTAC SGSETPGTSESATTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTG PESGPGTSTEPSEAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAA GSAPGTSTEPSEGGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGG SAPGTSTEPSEGSCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCA APGTSTEPSEGSAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGC PGTSTEPSEGSAPGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGC GTSTEPSEGSAPGACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGG SPAGSPTSTEEGTAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCA STEPSEGSAPGTSGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGG ESATPESGPGSEPTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTA ATSGSETPGTSESCCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGC ATPESGPGSEPATGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTC SGSETPGTSESATTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA PESGPGTSTEPSECTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAA GSAPGTSESATPEAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGG SGPGSPAGSPTSTCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCT EEGSPAGSPTSTECTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCT EGSPAGSPTSTEECCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAAC GTSESATPESGPGCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTG TSTEPSEGSAPGTAGGGCAGCGCACCAGGTACCTCTGAAAGCGCAACTCCT SESATPESGPGSEGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTC PATSGSETPGTSETGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAAT SATPESGPGSEPACTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAA TSGSETPGTSESAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGG TPESGPGTSTEPSCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCAC EGSAPGSPAGSPTCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAA STEEGTSESATPEGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGG SGPGSEPATSGSETAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTA TPGTSESATPESGCTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGC PGSPAGSPTSTEECCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCC GSPAGSPTSTEEGGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTAC TSTEPSEGSAPGTCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAA SESATPESGPGTSGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGC ESATPESGPGTSEGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGC SATPESGPGSEPATACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTT TSGSETPGSEPATCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCC SGSETPGSPAGSPGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCCGAC TSTEEGTSTEPSETTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAG GSAPGTSTEPSEGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGC SAPGSEPATSGSEAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGA TPGTSESATPESGAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTG PGTSTEPSEGSAPGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCA G CCAGGTTAA CTLA4-ATGGCAATGCATGTTGCACAGCCTGCAGTTGTTCTGGC 700 MAMHVAQPAVV 725 AE158-AAGCAGCCGTGGTATTGCCAGCTTTGTTTGTGAATATGC LASSRGIASFVCE CTLA4-AAGTCCGGGTAAAGCAACCGAAGTTCGTGTTACCGTTC YASPGKATEVRV AE864,TGAGACAGGCAGATAGCCAGGTTACCGAAGTTTGTGCA TVLRQADSQVTE AC391GCAACCTATATGATGGGTAATGAACTGACCTTTCTGGA VCAATYMMGNETGATAGCATTTGTACCGGCACCAGCAGCGGTAATCAGG LTFLDDSICTGTSTTAATCTGACCATTCAGGGTCTGCGTGCAATGGATACC SGNQVNLTIQGLGGTCTGTATATTTGTAAAGTGGAACTGATGTATCCGCCT RAMDTGLYICKVCCGTATTATCTGGGTATTGGTAATGGCACCCAGATTTAT ELMYPPPYYLGIGGTTATTGATCCGGAAGGCGCGCCAAGCACGGGAGGTAC NGTQIYVIDPEGATTCTGAAAGCGCTACTCCGGAGTCCGGTCCAGGTACCT PSTGGTSESATPECTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACTTCT SGPGTSTEPSEGSACTGAACCTTCTGAGGGTAGCGCTCCAGGTACTTCTGA APGTSTEPSEGSAAAGCGCTACTCCGGAGTCCGGTCCAGGTACCTCTACCG PGTSESATPESGPAACCGTCCGAAGGCAGCGCTCCAGGTACTTCTACTGAA GTSTEPSEGSAPGCCTTCTGAGGGTAGCGCTCCAGGTACCTCTGAAAGCGC TSTEPSEGSAPGTTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGT SESATPESGPGTSCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCC TEPSEGSAPGTSTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGA EPSEGSAPGTSTEGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCT PSEGSAPGSPAGSCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGT PTSTEEGTSTEPSAGCGCACCAGGTCCAGAACCAACGGGGCCGGCCATGC EGSAPGPEPTGPAATGTGGCCCAGCCAGCCGTGGTGCTGGCAAGTTCACGC MHVAQPAVVLAGGTATTGCATCATTTGTGTGCGAATATGCATCACCTGGT SSRGIASFVCEYAAAAGCCACAGAAGTGCGCGTAACAGTACTGCGTCAGGC SPGKATEVRVTVCGATTCACAGGTGACAGAAGTTTGCGCTGCCACATACA LRQADSQVTEVCTGATGGGCAACGAGCTGACATTCCTGGACGATTCAATT AATYMMGNELTTGTACTGGTACAAGCTCAGGCAATCAGGTGAACCTGAC FLDDSICTGTSSGAATCCAAGGCCTGAGAGCTATGGACACAGGCCTGTACA NQVNLTIQGLRATCTGCAAAGTTGAGCTGATGTACCCTCCGCCTTATTACT MDTGLYICKVELTAGGCATTGGCAACGGTACACAGATCTATGTGATCGAT MYPPPYYLGIGNCCTGAGGGAGGTAGCCCGGCTGGCTCTCCTACCTCTAC GTQIYVIDPEGGSTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTG PAGSPTSTEEGTSGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCT ESATPESGPGTSTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGA EPSEGSAPGSPAGAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAG SPTSTEEGTSTEPSGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGT EGSAPGTSTEPSEACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAG GSAPGTSESATPECGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCG SGPGSEPATSGSEAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCG TPGSEPATSGSETGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGA PGSPAGSPTSTEEAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCG GTSESATPESGPGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAA TSTEPSEGSAPGTCCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTC STEPSEGSAPGSPTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGT AGSPTSTEEGTSTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCT EPSEGSAPGTSTEGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCC PSEGSAPGTSESAGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAG TPESGPGTSTEPSGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAA EGSAPGTSESATPTCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGA ESGPGSEPATSGSGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCG ETPGTSTEPSEGSCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGCA APGTSTEPSEGSACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCC PGTSESATPESGPAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAG GTSESATPESGPGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGT SPAGSPTSTEEGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAG SESATPESGPGSECGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCT PATSGSETPGTSECTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCT SATPESGPGTSTEACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTAC PSEGSAPGTSTEPTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCG SEGSAPGTSTEPSAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAA EGSAPGTSTEPSECCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACC GSAPGTSTEPSEGTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGT SAPGTSTEPSEGSCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCT APGSPAGSPTSTEACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGA EGTSTEPSEGSAPGGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTG GTSESATPESGPGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCT SEPATSGSETPGTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATC SESATPESGPGSETGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAA PATSGSETPGTSECCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGC SATPESGPGTSTECCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACC PSEGSAPGTSESAAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAG TPESGPGSPAGSPGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGT TSTEEGSPAGSPTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAG STEEGSPAGSPTSCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTT TEEGTSESATPESCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCT GPGTSTEPSEGSAACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCTGA PGTSESATPESGPAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTG GSEPATSGSETPGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGC TSESATPESGPGSGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAAC EPATSGSETPGTSCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTA ESATPESGPGTSTCTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCG EPSEGSAPGSPAGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACC SPTSTEEGTSESATCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGA TPESGPGSEPATSATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTG GSETPGTSESATPAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCC ESGPGSPAGSPTSGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGA TEEGSPAGSPTSTGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAG EEGTSTEPSEGSAAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCA PGTSESATPESGPGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGG GTSESATPESGPGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTA TSESATPESGPGSCTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGC EPATSGSETPGSEGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGA PATSGSETPGSPAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAG GSPTSTEEGTSTECAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACT PSEGSAPGTSTEPGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGA SEGSAPGSEPATSACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAA GSETPGTSESATPCCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCT ESGPGTSTEPSEGACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCC SAPG GAGGGCAGCGCACCAGGTTAACTLA4- ATGGCAATGCATGTTGCACAGCCTGCAGTTGTTCTGGC 701 MAMHVAQPAVV 726 AE158-AAGCAGCCGTGGTATTGCCAGCTTTGTTTGTGAATATGC LASSRGIASFVCE CTLA4-AAGTCCGGGTAAAGCAACCGAAGTTCGTGTTACCGTTC YASPGKATEVRV AE864,TGAGACAGGCAGATAGCCAGGTTACCGAAGTTTGTGCA TVLRQADSQVTE AC392GCAACCTATATGATGGGTAATGAACTGACCTTTCTGGA VCAATYMMGNETGATAGCATTTGTACCGGCACCAGCAGCGGTAATCAGG LTFLDDSICTGTSTTAATCTGACCATTCAGGGTCTGCGTGCAATGGATACC SGNQVNLTIQGLGGTCTGTATATTTGTAAAGTGGAACTGATGTATCCGCCT RAMDTGLYICKVCCGTATTATCTGGGTATTGGTAATGGCACCCAGATTTAT ELMYPPPYYLGIGGTTATTGATCCGGAACCGTGTCCGGATAGCGGCGCGCC NGTQIYVIDPEPCAAGCACGGGAGGTACTTCTGAAAGCGCTACTCCGGAGT PDSGAPSTGGTSECCGGTCCAGGTACCTCTACCGAACCGTCCGAAGGCAGC SATPESGPGTSTEGCTCCAGGTACTTCTACTGAACCTTCTGAGGGTAGCGCT PSEGSAPGTSTEPCCAGGTACTTCTGAAAGCGCTACTCCGGAGTCCGGTCC SEGSAPGTSESATAGGTACCTCTACCGAACCGTCCGAAGGCAGCGCTCCAG PESGPGTSTEPSEGTACTTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGT GSAPGTSTEPSEGACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTAC SAPGTSESATPESCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTT GPGTSTEPSEGSACTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCT PGTSTEPSEGSAPACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGC GTSTEPSEGSAPGAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCG SPAGSPTSTEEGTAACCGTCCGAGGGTAGCGCACCAGGTCCAGAACCAAC STEPSEGSAPGPEGGGGCCGGCCATGCATGTGGCCCAGCCAGCCGTGGTGC PTGPAMHVAQPATGGCAAGTTCACGCGGTATTGCATCATTTGTGTGCGAAT VVLASSRGIASFVATGCATCACCTGGTAAAGCCACAGAAGTGCGCGTAACA CEYASPGKATEVGTACTGCGTCAGGCCGATTCACAGGTGACAGAAGTTTG RVTVLRQADSQVCGCTGCCACATACATGATGGGCAACGAGCTGACATTCC TEVCAATYMMGTGGACGATTCAATTTGTACTGGTACAAGCTCAGGCAAT NELTFLDDSICTGCAGGTGAACCTGACAATCCAAGGCCTGAGAGCTATGGA TSSGNQVNLTIQGCACAGGCCTGTACATCTGCAAAGTTGAGCTGATGTACC LRAMDTGLYICKCTCCGCCTTATTACTTAGGCATTGGCAACGGTACACAG VELMYPPPYYLGIATCTATGTGATCGATCCTGAACCTTGCCCTGATTCAGGA GNGTQIYVIDPEPGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGG CPDSGGSPAGSPTTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTAC STEEGTSESATPECTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCC SGPGTSTEPSEGSCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCT APGSPAGSPTSTEACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTAC EGTSTEPSEGSAPTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAA GTSTEPSEGSAPGGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCT TSESATPESGPGSACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTAC EPATSGSETPGSECTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTC PATSGSETPGSPACGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACC GSPTSTEEGTSESCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGA ATPESGPGTSTEPGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGG SEGSAPGTSTEPSGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCC EGSAPGSPAGSPTACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAG STEEGTSTEPSEGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCG SAPGTSTEPSEGSCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGT APGTSESATPESGCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACC PGTSTEPSEGSAPAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAG GTSESATPESGPGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGT SEPATSGSETPGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTAC STEPSEGSAPGTSTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTT TEPSEGSAPGTSECTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCT SATPESGPGTSESGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGC ATPESGPGSPAGSTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAA PTSTEEGTSESATGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCA PESGPGSEPATSGACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGC SETPGTSESATPETACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGT SGPGTSTEPSEGSCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCC APGTSTEPSEGSAGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGA PGTSTEPSEGSAPAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGG GTSTEPSEGSAPGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGT TSTEPSEGSAPGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAG STEPSEGSAPGSPCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCG AGSPTSTEEGTSTAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCA EPSEGSAPGTSESCCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCC ATPESGPGSEPATAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAG SGSETPGTSESATGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGT PESGPGSEPATSGAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTAC SETPGTSESATPECTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTC SGPGTSTEPSEGSTACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTG APGTSESATPESGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCT PGSPAGSPTSTEEGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGG GSPAGSPTSTEEGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGCT SPAGSPTSTEEGTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCA SESATPESGPGTSACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTC TEPSEGSAPGTSETGAGGGCAGCGCACCAGGTACCTCTGAAAGCGCAACTC SATPESGPGSEPACTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGC TSGSETPGTSESATCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGA TPESGPGSEPATSATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTG GSETPGTSESATPAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCT ESGPGTSTEPSEGGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGC SAPGSPAGSPTSTACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAG EEGTSESATPESGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCA PGSEPATSGSETPGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGG GTSESATPESGPGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTA SPAGSPTSTEEGSGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGC PAGSPTSTEEGTSCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCT TEPSEGSAPGTSEACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGA SATPESGPGTSESAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAA ATPESGPGTSESAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGC TPESGPGSEPATSGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTAC GSETPGSEPATSGTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCT SETPGSPAGSPTSCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCCG TEEGTSTEPSEGSACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGA APGTSTEPSEGSAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGG PGSEPATSGSETPGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCT GTSESATPESGPGGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATC TSTEPSEGSAPGTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCG CACCAGGTTAA AE912-ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGA 702 MAEPAGSPTSTEE 727 aIL6RAGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGG GTPGSGTASSSPG scFv,TAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGC SSTPSGATGSPGA AC341TTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCCC SPGTSSTGSPGSPGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGA AGSPTSTEEGTSEAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTG SATPESGPGTSTEAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGC PSEGSAPGSPAGSTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCT PTSTEEGTSTEPSTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTC EGSAPGTSTEPSETGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCC GSAPGTSESATPECGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGT SGPGSEPATSGSETCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTC TPGSEPATSGSETTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTA PGSPAGSPTSTEECTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCC GTSESATPESGPGGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGC TSTEPSEGSAPGTACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCAC STEPSEGSAPGSPCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAA AGSPTSTEEGTSTGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGG EPSEGSAPGTSTETACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA PSEGSAPGTSESACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACT TPESGPGTSTEPSTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTC EGSAPGTSESATPTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAAC ESGPGSEPATSGSCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCG ETPGTSTEPSEGSAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAA APGTSTEPSEGSACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGC PGTSESATPESGPAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAA GTSESATPESGPGCCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCA SPAGSPTSTEEGTACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCC SESATPESGPGSETGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTT PATSGSETPGTSECTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAG SATPESGPGTSTETCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAG PSEGSAPGTSTEPCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG SEGSAPGTSTEPSCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCT EGSAPGTSTEPSECCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCC GSAPGTSTEPSEGAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAG SAPGTSTEPSEGSGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT APGSPAGSPTSTEAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTAC EGTSTEPSEGSAPTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCT GTSESATPESGPGCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAA SEPATSGSETPGTCCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAA SESATPESGPGSEAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGC PATSGSETPGTSEAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCG SATPESGPGTSTECTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGT PSEGSAPGTSESACCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACT TPESGPGSPAGSPCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGAC TSTEEGSPAGSPTTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTT STEEGSPAGSPTSCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCT TEEGTSESATPESACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTC GPGTSTEPSEGSACGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCG PGTSESATPESGPCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGC GSEPATSGSETPGCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCC TSESATPESGPGSAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAG EPATSGSETPGTSGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT ESATPESGPGTSTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTAC EPSEGSAPGSPAGTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCC SPTSTEEGTSESACTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCT TPESGPGSEPATSGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACC GSETPGTSESATPGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAA ESGPGSPAGSPTSGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGC TEEGSPAGSPTSTTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTC EEGTSTEPSEGSATCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTC PGTSESATPESGPCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCC GTSESATPESGPGCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCT TSESATPESGPGSGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGA EPATSGSETPGSEATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTG PATSGSETPGSPAAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAA GSPTSTEEGTSTEACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGA PSEGSAPGTSTEPGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCAC SEGSAPGSEPATSCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA GSETPGTSESATPGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGG ESGPGTSTEPSEGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTA SAPGADIQMTQSCTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTGCT PSSLSASVGDRVTGATATTCAAATGACTCAATCTCCTTCTTCTCTGTCCGCA ITCRASQDISSYLTCTGTAGGCGACCGTGTAACCATCACTTGCCGTGCCTCC NWYQQKPGKAPCAGGACATCTCCAGCTACCTGAACTGGTACCAGCAGAA KLLIYYTSRLHSGGCCGGGCAAGGCTCCGAAACTGCTGATTTATTACACTA VPSRFSGSGSGTDGCCGTCTGCATTCTGGTGTTCCGAGCCGCTTCTCCGGTT FTFTISSLQPEDIACTGGCAGCGGTACCGATTTCACTTTTACTATCTCCAGCC TYYCQQGNTLPYTGCAACCGGAGGACATCGCGACGTACTATTGCCAGCAA TFGQGTKVEIKTGGTAATACCCTGCCGTACACCTTCGGCCAAGGCACGAA GSGEGSEGEGGGAGTTGAAATCAAAACCGGTTCTGGCGAAGGCTCTGAAG EGSEGEGSGEGGGTGAAGGTGGTGGTGAAGGCTCTGAAGGTGAAGGATCT EGEGSGSQVQLQGGTGAAGGTGGCGAAGGTGAGGGTTCTGGATCCCAAGT ESGPGLVRPSQTLTCAGCTGCAGGAATCTGGTCCGGGTCTGGTTCGTCCGTC SLTCTVSGYSITSTCAGACCCTGTCCCTGACCTGCACGGTGTCCGGCTACTC DHAWSWVRQPPTATTACCTCTGACCATGCGTGGTCCTGGGTCCGTCAGCC GRGLEWIGYISYSACCGGGTCGCGGTCTGGAGTGGATCGGCTACATCAGCT GITTYNPSLKSRVACAGCGGCATCACCACTTACAACCCGTCCCTGAAAAGC TMLRDTSKNQFSCGTGTCACCATGCTGCGTGACACCTCCAAAAATCAATT LRLSSVTAADTACTCCCTGCGCCTGAGCTCTGTGACGGCGGCCGACACTG VYYCARSLARTTCGGTGTACTACTGCGCTCGCAGCCTGGCGCGTACCACT AMDYWGQGSLVGCTATGGATTACTGGGGTCAGGGCAGCCTGGTAACCGT TVSS CAGCAGCTAA aIL6RATGGCTGATATTCAAATGACTCAATCTCCTTCTTCTCTG 703 MADIQMTQSPSS 728 scFv-TCCGCATCTGTAGGCGACCGTGTAACCATCACTTGCCGT LSASVGDRVTITC AE864,GCCTCCCAGGACATCTCCAGCTACCTGAACTGGTACCA RASQDISSYLNW AC342GCAGAAGCCGGGCAAGGCTCCGAAACTGCTGATTTATT YQQKPGKAPKLLACACTAGCCGTCTGCATTCTGGTGTTCCGAGCCGCTTCT IYYTSRLHSGVPSCCGGTTCTGGCAGCGGTACCGATTTCACTTTTACTATCT RFSGSGSGTDFTFCCAGCCTGCAACCGGAGGACATCGCGACGTACTATTGC TISSLQPEDIATYCAGCAAGGTAATACCCTGCCGTACACCTTCGGCCAAGG YCQQGNTLPYTFCACGAAAGTTGAAATCAAAACCGGTTCTGGCGAAGGCT GQGTKVEIKTGSCTGAAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGTGA GEGSEGEGGGEGAGGATCTGGTGAAGGTGGCGAAGGTGAGGGTTCTGGAT SEGEGSGEGGEGCCCAAGTTCAGCTGCAGGAATCTGGTCCGGGTCTGGTT EGSGSQVQLQESCGTCCGTCTCAGACCCTGTCCCTGACCTGCACGGTGTCC GPGLVRPSQTLSLGGCTACTCTATTACCTCTGACCATGCGTGGTCCTGGGTC TCTVSGYSITSDHCGTCAGCCACCGGGTCGCGGTCTGGAGTGGATCGGCTA AWSWVRQPPGRCATCAGCTACAGCGGCATCACCACTTACAACCCGTCCC GLEWIGYISYSGITGAAAAGCCGTGTCACCATGCTGCGTGACACCTCCAAA TTYNPSLKSRVTAATCAATTCTCCCTGCGCCTGAGCTCTGTGACGGCGGC MLRDTSKNQFSLCGACACTGCGGTGTACTACTGCGCTCGCAGCCTGGCGC RLSSVTAADTAVGTACCACTGCTATGGATTACTGGGGTCAGGGCAGCCTG YYCARSLARTTAGTAACCGTCAGCAGCGGGTCTCCAGGTAGCCCGGCTGG MDYWGQGSLVTCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCG VSSGSPGSPAGSPCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGT TSTEEGTSESATPCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTCCG ESGPGTSTEPSEGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGA SAPGSPAGSPTSTAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGG EEGTSTEPSEGSAGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAA PGTSTEPSEGSAPTCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGA GTSESATPESGPGAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAA SEPATSGSETPGSCTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAG EPATSGSETPGSPGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCC AGSPTSTEEGTSEAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAG SATPESGPGTSTEGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT PSEGSAPGTSTEPAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTAC SEGSAPGSPAGSPTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCT TSTEEGTSTEPSECTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCT GSAPGTSTEPSEGGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTAC SAPGTSESATPESTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAA GPGTSTEPSEGSAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCT PGTSESATPESGPACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCG GSEPATSGSETPGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTC TSTEPSEGSAPGTTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCC STEPSEGSAPGTSCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCG ESATPESGPGTSEGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTC SATPESGPGSPAGCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAAT SPTSTEEGTSESACCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAA TPESGPGSEPATSACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGG GSETPGTSESATPCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTC ESGPGTSTEPSEGCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA SAPGTSTEPSEGSGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGG APGTSTEPSEGSATACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTA PGTSTEPSEGSAPCCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACT GTSTEPSEGSAPGTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCC TSTEPSEGSAPGSAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTA PAGSPTSTEEGTSCCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAA TEPSEGSAPGTSEAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGC SATPESGPGSEPATACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCG TSGSETPGTSESACAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACC TPESGPGSEPATSTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTAC GSETPGTSESATPTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGA ESGPGTSTEPSEGGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCTG SAPGTSESATPESAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCC GPGSPAGSPTSTEACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTAC EGSPAGSPTSTEETGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACTG GSPAGSPTSTEEGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGC TSESATPESGPGTCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACC STEPSEGSAPGTSAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAG ESATPESGPGSEPGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGT ATSGSETPGTSESACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAG ATPESGPGSEPATCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCT SGSETPGTSESATCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCT PESGPGTSTEPSEACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGC GSAPGSPAGSPTSTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAA TEEGTSESATPESGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCA GPGSEPATSGSETACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGC PGTSESATPESGPTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTC GSPAGSPTSTEEGCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCA SPAGSPTSTEEGTACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCCGA STEPSEGSAPGTSGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTG ESATPESGPGTSEAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAA SATPESGPGTSESTCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATC ATPESGPGSEPATTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAA SGSETPGSEPATSCCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACT GSETPGSPAGSPTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGA STEEGTSTEPSEGAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAG SAPGTSTEPSEGSGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGT APGSEPATSGSETAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTAC PGTSESATPESGPCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTC GTSTEPSEGSAPGTACTGAACCGTCCGAGGGCAGCGCACCAGGTTAA AE912-ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGA 704 MAEPAGSPTSTEE 729 aIL6R-AGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGG GTPGSGTASSSPG AE144,TAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGC SSTPSGATGSPGA AC361TTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCCC SPGTSSTGSPGSPGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGA AGSPTSTEEGTSEAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTG SATPESGPGTSTEAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGC PSEGSAPGSPAGSTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCT PTSTEEGTSTEPSTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTC EGSAPGTSTEPSETGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCC GSAPGTSESATPECGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGT SGPGSEPATSGSETCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTC TPGSEPATSGSETTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTA PGSPAGSPTSTEECTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCC GTSESATPESGPGGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGC TSTEPSEGSAPGTACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCAC STEPSEGSAPGSPCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAA AGSPTSTEEGTSTGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGG EPSEGSAPGTSTETACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA PSEGSAPGTSESACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACT TPESGPGTSTEPSTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTC EGSAPGTSESATPTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAAC ESGPGSEPATSGSCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCG ETPGTSTEPSEGSAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAA APGTSTEPSEGSACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGC PGTSESATPESGPAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAA GTSESATPESGPGCCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCA SPAGSPTSTEEGTACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCC SESATPESGPGSETGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTT PATSGSETPGTSECTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAG SATPESGPGTSTETCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAG PSEGSAPGTSTEPCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG SEGSAPGTSTEPSCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCT EGSAPGTSTEPSECCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCC GSAPGTSTEPSEGAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAG SAPGTSTEPSEGSGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT APGSPAGSPTSTEAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTAC EGTSTEPSEGSAPTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCT GTSESATPESGPGCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAA SEPATSGSETPGTCCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAA SESATPESGPGSEAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGC PATSGSETPGTSEAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCG SATPESGPGTSTECTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGT PSEGSAPGTSESACCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACT TPESGPGSPAGSPCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGAC TSTEEGSPAGSPTTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTT STEEGSPAGSPTSCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCT TEEGTSESATPESACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTC GPGTSTEPSEGSACGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCG PGTSESATPESGPCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGC GSEPATSGSETPGCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCC TSESATPESGPGSAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAG EPATSGSETPGTSGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT ESATPESGPGTSTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTAC EPSEGSAPGSPAGTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCC SPTSTEEGTSESACTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCT TPESGPGSEPATSGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACC GSETPGTSESATPGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAA ESGPGSPAGSPTSGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGC TEEGSPAGSPTSTTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTC EEGTSTEPSEGSATCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTC PGTSESATPESGPCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCC GTSESATPESGPGCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCT TSESATPESGPGSGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGA EPATSGSETPGSEATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTG PATSGSETPGSPAAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAA GSPTSTEEGTSTEACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGA PSEGSAPGTSTEPGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCAC SEGSAPGSEPATSCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA GSETPGTSESATPGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGG ESGPGTSTEPSEGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTA SAPGADIQMTQSCTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTGCT PSSLSASVGDRVTGATATTCAAATGACTCAATCTCCTTCTTCTCTGTCCGCA ITCRASQDISSYLTCTGTAGGCGACCGTGTAACCATCACTTGCCGTGCCTCC NWYQQKPGKAPCAGGACATCTCCAGCTACCTGAACTGGTACCAGCAGAA KLLIYYTSRLHSGGCCGGGCAAGGCTCCGAAACTGCTGATTTATTACACTA VPSRFSGSGSGTDGCCGTCTGCATTCTGGTGTTCCGAGCCGCTTCTCCGGTT FTFTISSLQPEDIACTGGCAGCGGTACCGATTTCACTTTTACTATCTCCAGCC TYYCQQGNTLPYTGCAACCGGAGGACATCGCGACGTACTATTGCCAGCAA TFGQGTKVEIKTGGTAATACCCTGCCGTACACCTTCGGCCAAGGCACGAA GSGEGSEGEGGGAGTTGAAATCAAAACCGGTTCTGGCGAAGGCTCTGAAG EGSEGEGSGEGGGTGAAGGTGGTGGTGAAGGCTCTGAAGGTGAAGGATCT EGEGSGSQVQLQGGTGAAGGTGGCGAAGGTGAGGGTTCTGGATCCCAAGT ESGPGLVRPSQTLTCAGCTGCAGGAATCTGGTCCGGGTCTGGTTCGTCCGTC SLTCTVSGYSITSTCAGACCCTGTCCCTGACCTGCACGGTGTCCGGCTACTC DHAWSWVRQPPTATTACCTCTGACCATGCGTGGTCCTGGGTCCGTCAGCC GRGLEWIGYISYSACCGGGTCGCGGTCTGGAGTGGATCGGCTACATCAGCT GITTYNPSLKSRVACAGCGGCATCACCACTTACAACCCGTCCCTGAAAAGC TMLRDTSKNQFSCGTGTCACCATGCTGCGTGACACCTCCAAAAATCAATT LRLSSVTAADTACTCCCTGCGCCTGAGCTCTGTGACGGCGGCCGACACTG VYYCARSLARTTCGGTGTACTACTGCGCTCGCAGCCTGGCGCGTACCACT AMDYWGQGSLVGCTATGGATTACTGGGGTCAGGGCAGCCTGGTAACCGT TVSSGGTSESATPCAGCAGCGGAGGTACTTCTGAAAGCGCTACTCCGGAGT ESGPGTSTEPSEGCCGGTCCAGGTACCTCTACCGAACCGTCCGAAGGCAGC SAPGTSTEPSEGSGCTCCAGGTACTTCTACTGAACCTTCTGAGGGTAGCGCT APGTSESATPESGCCAGGTACTTCTGAAAGCGCTACTCCGGAGTCCGGTCC PGTSTEPSEGSAPAGGTACCTCTACCGAACCGTCCGAAGGCAGCGCTCCAG GTSTEPSEGSAPGGTACTTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGT TSESATPESGPGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTAC STEPSEGSAPGTSCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTT TEPSEGSAPGTSTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCT EPSEGSAPGSPAGACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGC SPTSTEEGTSTEPSAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCG EGSAPGAACCGTCCGAGGGTAGCGCACCAGGTTAA AE48-ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGA 705 MAEPAGSPTSTEE 730 aIL6R-AGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGG GTPGSGTASSSPG AE864,TAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGC SSTPSGATGSPGA AC362TTCTCCGGGCACCAGCTCTACCGGTTCTCCAgggtctccaggt SPGTSSTGSPGSPGATATTCAAATGACTCAATCTCCTTCTTCTCTGTCCGCA GDIQMTQSPSSLSTCTGTAGGCGACCGTGTAACCATCACTTGCCGTGCCTCC ASVGDRVTITCRCAGGACATCTCCAGCTACCTGAACTGGTACCAGCAGAA ASQDISSYLNWYGCCGGGCAAGGCTCCGAAACTGCTGATTTATTACACTA QQKPGKAPKLLIGCCGTCTGCATTCTGGTGTTCCGAGCCGCTTCTCCGGTT YYTSRLHSGVPSCTGGCAGCGGTACCGATTTCACTTTTACTATCTCCAGCC RFSGSGSGTDFTFTGCAACCGGAGGACATCGCGACGTACTATTGCCAGCAA TISSLQPEDIATYGGTAATACCCTGCCGTACACCTTCGGCCAAGGCACGAA YCQQGNTLPYTFAGTTGAAATCAAAACCGGTTCTGGCGAAGGCTCTGAAG GQGTKVEIKTGSGTGAAGGTGGTGGTGAAGGCTCTGAAGGTGAAGGATCT GEGSEGEGGGEGGGTGAAGGTGGCGAAGGTGAGGGTTCTGGATCCCAAGT SEGEGSGEGGEGTCAGCTGCAGGAATCTGGTCCGGGTCTGGTTCGTCCGTC EGSGSQVQLQESTCAGACCCTGTCCCTGACCTGCACGGTGTCCGGCTACTC GPGLVRPSQTLSLTATTACCTCTGACCATGCGTGGTCCTGGGTCCGTCAGCC TCTVSGYSITSDHACCGGGTCGCGGTCTGGAGTGGATCGGCTACATCAGCT AWSWVRQPPGRACAGCGGCATCACCACTTACAACCCGTCCCTGAAAAGC GLEWIGYISYSGICGTGTCACCATGCTGCGTGACACCTCCAAAAATCAATT TTYNPSLKSRVTCTCCCTGCGCCTGAGCTCTGTGACGGCGGCCGACACTG MLRDTSKNQFSLCGGTGTACTACTGCGCTCGCAGCCTGGCGCGTACCACT RLSSVTAADTAVGCTATGGATTACTGGGGTCAGGGCAGCCTGGTAACCGT YYCARSLARTTACAGCAGCGGAGGTAGCCCGGCTGGCTCTCCTACCTCTA MDYWGQGSLVTCTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCT VSSGGSPAGSPTSGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGC TEEGTSESATPESTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGG GPGTSTEPSEGSAAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCA PGSPAGSPTSTEEGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGG GTSTEPSEGSAPGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTA TSTEPSEGSAPGTGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGC SESATPESGPGSEGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCC PATSGSETPGSEPGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTG ATSGSETPGSPAGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACC SPTSTEEGTSESAGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGA TPESGPGTSTEPSACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTT EGSAPGTSTEPSECTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCG GSAPGSPAGSPTSTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTC TEEGTSTEPSEGSTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCC APGTSTEPSEGSACGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAA PGTSESATPESGPGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGA GTSTEPSEGSAPGATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTG TSESATPESGPGSAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGC EPATSGSETPGTSGCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGC TEPSEGSAPGTSTACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCC EPSEGSAPGTSESCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCA ATPESGPGTSESAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGG TPESGPGSPAGSPTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTA TSTEEGTSESATPGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACC ESGPGSEPATSGSTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTC ETPGTSESATPESTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTA GPGTSTEPSEGSACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACC PGTSTEPSEGSAPGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGA GTSTEPSEGSAPGACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAAC TSTEPSEGSAPGTCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCG STEPSEGSAPGTSTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCC TEPSEGSAPGSPATACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCG GSPTSTEEGTSTEAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCT PSEGSAPGTSESAGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTC TPESGPGSEPATSTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAAT GSETPGTSESATPCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAA ESGPGSEPATSGSACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGG ETPGTSESATPESCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCAC GPGTSTEPSEGSACAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCA PGTSESATPESGPGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGG GSPAGSPTSTEEGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTA SPAGSPTSTEEGSGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACT PAGSPTSTEEGTSTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTC ESATPESGPGTSTTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCTG EPSEGSAPGTSESAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCT ATPESGPGSEPATGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAG SGSETPGTSESATCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAA PESGPGSEPATSGCCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCT SETPGTSESATPEACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCC SGPGTSTEPSEGSGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAAC APGSPAGSPTSTECTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTG EGTSESATPESGPAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCT GSEPATSGSETPGGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTC TSESATPESGPGSCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCG PAGSPTSTEEGSPAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAA AGSPTSTEEGTSTGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACC EPSEGSAPGTSESAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAG ATPESGPGTSESAGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGT TPESGPGTSESATACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAG PESGPGSEPATSGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCG SETPGSEPATSGSAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCA ETPGSPAGSPTSTGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTAC EEGTSTEPSEGSATGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTG PGTSTEPSEGSAPAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCA GSEPATSGSETPGACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGC TSESATPESGPGTTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTC STEPSEGSAPGCGAGGGCAGCGCACCAGGTTAA anti- ATGGCTGAAATTGTTCTGACCCAATCTCCTGCAACTCTG 706MAEIVLTQSPATL 731 CD40- TCTCTGTCTCCAGGTGAACGCGCCACCCTGTCTTGTCGTSLSPGERATLSCR AE864, GCGTCCCAGTCTATCTCTGATTATCTGCATTGGTATCAGASQSISDYLHWY AC384 CAGAAACCTGGCCAGGCTCCGCGCCTGCTGATCTATTA QQKPGQAPRLLICGCCAGCCACAGCATCTCTGGTATCCCGGCTCGCTTCTC YYASHSISGIPARCGGCTCCGGCAGCGGCACCGACTTCACTCTGACTATTA FSGSGSGTDFTLTGCTCCCTGGAACCGGAGGATTTCGCAGTTTATTACTGTC ISSLEPEDFAVYYAGCACGGTCACTCCTACCCGTGGACCTTTGGTGGCGGC CQHGHSYPWTFGACCAAAGTTGAAATCAAAACCGGTTCTGGCGAAGGCTC GGTKVEIKTGSGTGAAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGTGAA EGSEGEGGGEGSGGATCTGGTGAAGGTGGCGAAGGTGAGGGATCTGGTAC EGEGSGEGGEGECCAAGTCCAGCTGGTTCAGTCCGGCTCTGAACTGAAGA GSGTQVQLVQSGAACCGGGCGCTTCTGTTAAAGTTAGCTGCAAAGCAAGC SELKKPGASVKVGGTTATGCCTTTACTACTACTGGTATGCAGTGGGTCCGC SCKASGYAFTTTCAGGCACCGGGTCAGGGCCTGGAGTGGATGGGCTGGAT GMQWVRQAPGQCAACACCCACTCTGGTGTCCCTAAATACGTTGAAGATTT GLEWMGWINTHCAAAGGCCGTTTCGTGTTCTCCCTGGACACTTCCGTCAG SGVPKYVEDFKGCACCGCGTATCTGCAGATCAGCAGCCTGAAAGCTGAGG RFVFSLDTSVSTAACACCGCGGTTTATTACTGCGCGCGTAGCGGCAATGGT YLQISSLKAEDTAAACTACGACCTGGCTTATTTCAAATACTGGGGTCAGGG VYYCARSGNGNCACTCTGGTTACTGTGTCTAGCGGAGGTAGCCCGGCTG YDLAYFKYWGQGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGC GTLVTVSSGGSPGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCG AGSPTSTEEGTSETCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTCC SATPESGPGTSTEGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCG PSEGSAPGSPAGSAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAG PTSTEEGTSTEPSGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGA EGSAPGTSTEPSEATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTG GSAPGTSESATPEAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAA SGPGSEPATSGSEACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGA TPGSEPATSGSETGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCC PGSPAGSPTSTEECAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA GTSESATPESGPGGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGG TSTEPSEGSAPGTTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTA STEPSEGSAPGSPCTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACC AGSPTSTEEGTSTTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCT EPSEGSAPGTSTEGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTAC PSEGSAPGTSESATGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAA TPESGPGTSTEPSGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCT EGSAPGTSESATPACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCG ESGPGSEPATSGSTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTC ETPGTSTEPSEGSTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCC APGTSTEPSEGSACGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCG PGTSESATPESGPGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTC GTSESATPESGPGCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAAT SPAGSPTSTEEGTCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAA SESATPESGPGSEACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGG PATSGSETPGTSECCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTC SATPESGPGTSTECAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA PSEGSAPGTSTEPGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGG SEGSAPGTSTEPSTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTA EGSAPGTSTEPSECCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACT GSAPGTSTEPSEGTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCC SAPGTSTEPSEGSAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTA APGSPAGSPTSTECCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAA EGTSTEPSEGSAPAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGC GTSESATPESGPGTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCG SEPATSGSETPGTCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACC SESATPESGPGSETCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTAC PATSGSETPGTSETCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGA SATPESGPGTSTEGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCTG PSEGSAPGTSESAAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCC TPESGPGSPAGSPACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTAC TSTEEGSPAGSPTTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACTG STEEGSPAGSPTSAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGC TEEGTSESATPESCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACC GPGTSTEPSEGSAAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAG PGTSESATPESGPGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGT GSEPATSGSETPGACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAG TSESATPESGPGSCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCT EPATSGSETPGTSCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCT ESATPESGPGTSTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGC EPSEGSAPGSPAGTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAA SPTSTEEGTSESAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCA TPESGPGSEPATSACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGC GSETPGTSESATPTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTC ESGPGSPAGSPTSCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCA TEEGSPAGSPTSTACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCCGA EEGTSTEPSEGSAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTG PGTSESATPESGPAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAA GTSESATPESGPGTCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATC TSESATPESGPGSTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAA EPATSGSETPGSECCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACT PATSGSETPGSPACCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGA GSPTSTEEGTSTEAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAG PSEGSAPGTSTEPGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGT SEGSAPGSEPATSAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTAC GSETPGTSESATPCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTC ESGPGTSTEPSEGTACTGAACCGTCCGAGGGCAGCGCACCA SAP anti-ATGGCTGAAATTGTTCTGACTCAATCTCCAGCAACTCTG 707 MAEIVLTQSPATL 732 CD40-TCTCTGTCTCCAGGTGAACGTGCAACCCTGTCTTGCCGT SLSPGERATLSCR AE864,GCGTCCCAGTCCATCTCCGATTATCTGCATTGGTATCAG ASQSISDYLHWY AC385CAGAAACCGGGTCAGGCGCCTCGTCTGCTGATCTATTA QQKPGQAPRLLITGCGTCTCACTCCATTTCCGGTATCCCGGCACGTTTCTC YYASHSISGIPARTGGCAGCGGCAGCGGCACCGATTTCACCCTGACGATCT FSGSGSGTDFTLTCTTCTCTGGAACCGGAAGATTTCGCAGTCTATTATTGTC ISSLEPEDFAVYYAGCATGGTCACAGCTACCCGTGGACCTTCGGCGGTGGC CQHGHSYPWTFGACGAAAGTTGAAATCAAGACCGGTTCTGGCGAAGGCTC GGTKVEIKTGSGTGAAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGTGAA EGSEGEGGGEGSGGATCTGGTGAAGGTGGCGAAGGTGAGGGATCTGGTAC EGEGSGEGGEGECCAGGTCCAGCTGGTTCAAAGCGGCTCTGAACTGAAAA GSGTQVQLVQSGAGCCGGGTGCCTCTGTCAAAGTGTCTTGCAAGGCAAGC SELKKPGASVKVGGCTACGCGTTTACGACCACCGGCATGCAGTGGGTCCG SCKASGYAFTTTTCAGGCCCCGGGCCAGGGTCTGGAATGGATGGGCTGGA GMQWVRQAPGQTCAACACCCATTCTGGCGTACCGAAATACGTTGAAGAT GLEWMGWINTHTTCAAAGGCCGTTTCGTGTTCTCCCTGGATACGTCCGTT SGVPKYVEDFKGTCCACCGCCTACCTGCAGATCTCTTCCCTGAAAGCAGA RFVFSLDTSVSTAAGATACTGCGGTGTACTATTGCGCACGTAGCGGCAACG YLQISSLKAEDTAGCAACTACGACCTGGCCTACTTCAAATACTGGGGTCAG VYYCARSGNGNGGTACTCTGGTGACCGTATCCTCTGGAGGTAGCCCGGC YDLAYFKYWGQTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAA GTLVTVSSGGSPGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAAC AGSPTSTEEGTSECGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCT SATPESGPGTSTECCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCC PSEGSAPGSPAGSGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGA PTSTEEGTSTEPSGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGG EGSAPGTSTEPSEAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCT GSAPGTSESATPEGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGA SGPGSEPATSGSEAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTG TPGSEPATSGSETAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGC PGSPAGSPTSTEECCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACC GTSESATPESGPGAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAG TSTEPSEGSAPGTGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGT STEPSEGSAPGSPACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAC AGSPTSTEEGTSTCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTC EPSEGSAPGTSTETGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTA PSEGSAPGTSESACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAA TPESGPGTSTEPSAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGC EGSAPGTSESATPTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACC ESGPGSEPATSGSGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGT ETPGTSTEPSEGSCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACC APGTSTEPSEGSACCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCC PGTSESATPESGPGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCT GTSESATPESGPGCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAA SPAGSPTSTEEGTTCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGA SESATPESGPGSEAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTG PATSGSETPGTSEGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCT SATPESGPGTSTECCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACC PSEGSAPGTSTEPAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAG SEGSAPGTSTEPSGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGT EGSAPGTSTEPSEACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTAC GSAPGTSTEPSEGTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCC SAPGTSTEPSEGSCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCT APGSPAGSPTSTEACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGA EGTSTEPSEGSAPAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTG GTSESATPESGPGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGC SEPATSGSETPGTGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAAC SESATPESGPGSECTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTA PATSGSETPGTSECTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCG SATPESGPGTSTEAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCT PSEGSAPGTSESAGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTC TPESGPGSPAGSPCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTA TSTEEGSPAGSPTCTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACT STEEGSPAGSPTSGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGG TEEGTSESATPESCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCAC GPGTSTEPSEGSACAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCA PGTSESATPESGPGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGG GSEPATSGSETPGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTA TSESATPESGPGSGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC EPATSGSETPGTSTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCT ESATPESGPGTSTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGC EPSEGSAPGSPAGTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAA SPTSTEEGTSESAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCA TPESGPGSEPATSACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGC GSETPGTSESATPTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTC ESGPGSPAGSPTSCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCA TEEGSPAGSPTSTACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCCGA EEGTSTEPSEGSAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTG PGTSESATPESGPAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAA GTSESATPESGPGTCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATC TSESATPESGPGSTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAA EPATSGSETPGSECCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACT PATSGSETPGSPACCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGA GSPTSTEEGTSTEAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAG PSEGSAPGTSTEPGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGT SEGSAPGSEPATSAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTAC GSETPGTSESATPCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTC ESGPGTSTEPSEGTACTGAACCGTCCGAGGGCAGCGCACCA SAP AE912-ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGA 708 MAEPAGSPTSTEE 733 anti-AGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGG GTPGSGTASSSPG CD40,TAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGC SSTPSGATGSPGA AC386TTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCCC SPGTSSTGSPGSPGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGA AGSPTSTEEGTSEAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTG SATPESGPGTSTEAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGC PSEGSAPGSPAGSTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCT PTSTEEGTSTEPSTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTC EGSAPGTSTEPSETGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCC GSAPGTSESATPECGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGT SGPGSEPATSGSETCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTC TPGSEPATSGSETTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTA PGSPAGSPTSTEECTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCC GTSESATPESGPGGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGC TSTEPSEGSAPGTACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCAC STEPSEGSAPGSPCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAA AGSPTSTEEGTSTGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGG EPSEGSAPGTSTETACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA PSEGSAPGTSESACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACT TPESGPGTSTEPSTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTC EGSAPGTSESATPTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAAC ESGPGSEPATSGSCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCG ETPGTSTEPSEGSAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAA APGTSTEPSEGSACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGC PGTSESATPESGPAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAA GTSESATPESGPGCCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCA SPAGSPTSTEEGTACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCC SESATPESGPGSETGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTT PATSGSETPGTSECTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAG SATPESGPGTSTETCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAG PSEGSAPGTSTEPCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG SEGSAPGTSTEPSCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCT EGSAPGTSTEPSECCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCC GSAPGTSTEPSEGAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAG SAPGTSTEPSEGSGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT APGSPAGSPTSTEAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTAC EGTSTEPSEGSAPTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCT GTSESATPESGPGCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAA SEPATSGSETPGTCCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAA SESATPESGPGSEAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGC PATSGSETPGTSEAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCG SATPESGPGTSTECTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGT PSEGSAPGTSESACCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACT TPESGPGSPAGSPCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGAC TSTEEGSPAGSPTTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTT STEEGSPAGSPTSCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCT TEEGTSESATPESACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTC GPGTSTEPSEGSACGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCG PGTSESATPESGPCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGC GSEPATSGSETPGCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCC TSESATPESGPGSAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAG EPATSGSETPGTSGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT ESATPESGPGTSTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTAC EPSEGSAPGSPAGTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCC SPTSTEEGTSESACTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCT TPESGPGSEPATSGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACC GSETPGTSESATPGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAA ESGPGSPAGSPTSGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGC TEEGSPAGSPTSTTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTC EEGTSTEPSEGSATCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTC PGTSESATPESGPCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCC GTSESATPESGPGCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCT TSESATPESGPGSGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGA EPATSGSETPGSEATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTG PATSGSETPGSPAAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAA GSPTSTEEGTSTEACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGA PSEGSAPGTSTEPGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCAC SEGSAPGSEPATSCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA GSETPGTSESATPGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGG ESGPGTSTEPSEGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTA SAPGEIVLTQSPACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTGAA TLSLSPGERATLSATTGTTCTGACCCAATCTCCTGCAACTCTGTCTCTGTCT CRASQSISDYLHCCAGGTGAACGCGCCACCCTGTCTTGTCGTGCGTCCCA WYQQKPGQAPRGTCTATCTCTGATTATCTGCATTGGTATCAGCAGAAACC LLIYYASHSISGIPTGGCCAGGCTCCGCGCCTGCTGATCTATTACGCCAGCC ARFSGSGSGTDFTACAGCATCTCTGGTATCCCGGCTCGCTTCTCCGGCTCCG LTISSLEPEDFAVGCAGCGGCACCGACTTCACTCTGACTATTAGCTCCCTG YYCQHGHSYPWGAACCGGAGGATTTCGCAGTTTATTACTGTCAGCACGG TFGGGTKVEIKTTCACTCCTACCCGTGGACCTTTGGTGGCGGCACCAAAG GSGEGSEGEGGGTTGAAATCAAAACCGGTTCTGGCGAAGGCTCTGAAGGT EGSEGEGSGEGGGAAGGTGGTGGTGAAGGCTCTGAAGGTGAAGGATCTG EGEGSGTQVQLVGTGAAGGTGGCGAAGGTGAGGGATCTGGTACCCAAGTC QSGSELKKPGASCAGCTGGTTCAGTCCGGCTCTGAACTGAAGAAACCGGG VKVSCKASGYAFCGCTTCTGTTAAAGTTAGCTGCAAAGCAAGCGGTTATG TTTGMQWVRQACCTTTACTACTACTGGTATGCAGTGGGTCCGCCAGGCA PGQGLEWMGWICCGGGTCAGGGCCTGGAGTGGATGGGCTGGATCAACAC NTHSGVPKYVEDCCACTCTGGTGTCCCTAAATACGTTGAAGATTTCAAAG FKGRFVFSLDTSVGCCGTTTCGTGTTCTCCCTGGACACTTCCGTCAGCACCG STAYLQISSLKAECGTATCTGCAGATCAGCAGCCTGAAAGCTGAGGACACC DTAVYYCARSGNGCGGTTTATTACTGCGCGCGTAGCGGCAATGGTAACTA GNYDLAYFKYWCGACCTGGCTTATTTCAAATACTGGGGTCAGGGCACTC GQGTLVTVS TGGTTACTGTGTCTAGCAE912- ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGA 709 MAEPAGSPTSTEE  734anti- AGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGG GTPGSGTASSSPG CD40,TAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGC SSTPSGATGSPGA AC387,TTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCCC SPGTSSTGSPGSPGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGA AGSPTSTEEGTSEAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTG SATPESGPGTSTEAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGC PSEGSAPGSPAGSTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCT PTSTEEGTSTEPSTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTC EGSAPGTSTEPSETGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCC GSAPGTSESATPECGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGT SGPGSEPATSGSETCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTC TPGSEPATSGSETTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTA PGSPAGSPTSTEECTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCC GTSESATPESGPGGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGC TSTEPSEGSAPGTACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCAC STEPSEGSAPGSPCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAA AGSPTSTEEGTSTGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGG EPSEGSAPGTSTETACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA PSEGSAPGTSESACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACT TPESGPGTSTEPSTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTC EGSAPGTSESATPTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAAC ESGPGSEPATSGSCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCG ETPGTSTEPSEGSAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAA APGTSTEPSEGSACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGC PGTSESATPESGPAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAA GTSESATPESGPGCCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCA SPAGSPTSTEEGTACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCC SESATPESGPGSETGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTT PATSGSETPGTSECTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAG SATPESGPGTSTETCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAG PSEGSAPGTSTEPCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG SEGSAPGTSTEPSCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCT EGSAPGTSTEPSECCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCC GSAPGTSTEPSEGAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAG SAPGTSTEPSEGSGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT APGSPAGSPTSTEAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTAC EGTSTEPSEGSAPTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCT GTSESATPESGPGCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAA SEPATSGSETPGTCCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAA SESATPESGPGSEAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGC PATSGSETPGTSEAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCG SATPESGPGTSTECTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGT PSEGSAPGTSESACCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACT TPESGPGSPAGSPCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGAC TSTEEGSPAGSPTTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTT STEEGSPAGSPTSCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCT TEEGTSESATPESACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTC GPGTSTEPSEGSACGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCG PGTSESATPESGPCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGC GSEPATSGSETPGCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCC TSESATPESGPGSAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAG EPATSGSETPGTSGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT ESATPESGPGTSTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTAC EPSEGSAPGSPAGTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCC SPTSTEEGTSESACTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCT TPESGPGSEPATSGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACC GSETPGTSESATPGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAA ESGPGSPAGSPTSGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGC TEEGSPAGSPTSTTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTC EEGTSTEPSEGSATCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTC PGTSESATPESGPCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCC GTSESATPESGPGCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCT TSESATPESGPGSGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGA EPATSGSETPGSEATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTG PATSGSETPGSPAAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAA GSPTSTEEGTSTEACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGA PSEGSAPGTSTEPGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCAC SEGSAPGSEPATSCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA GSETPGTSESATPGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGG ESGPGTSTEPSEGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTA SAPGEIVLTQSPACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTGAA TLSLSPGERATLSATTGTTCTGACTCAATCTCCAGCAACTCTGTCTCTGTCT CRASQSISDYLHCCAGGTGAACGTGCAACCCTGTCTTGCCGTGCGTCCCA WYQQKPGQAPRGTCCATCTCCGATTATCTGCATTGGTATCAGCAGAAACC LLIYYASHSISGIPGGGTCAGGCGCCTCGTCTGCTGATCTATTATGCGTCTCA ARFSGSGSGTDFTCTCCATTTCCGGTATCCCGGCACGTTTCTCTGGCAGCGG LTISSLEPEDFAVCAGCGGCACCGATTTCACCCTGACGATCTCTTCTCTGGA YYCQHGHSYPWACCGGAAGATTTCGCAGTCTATTATTGTCAGCATGGTC TFGGGTKVEIKTACAGCTACCCGTGGACCTTCGGCGGTGGCACGAAAGTT GSGEGSEGEGGGGAAATCAAGACCGGTTCTGGCGAAGGCTCTGAAGGTGA EGSEGEGSGEGGAGGTGGTGGTGAAGGCTCTGAAGGTGAAGGATCTGGTG EGEGSGTQVQLVAAGGTGGCGAAGGTGAGGGATCTGGTACCCAGGTCCA QSGSELKKPGASGCTGGTTCAAAGCGGCTCTGAACTGAAAAAGCCGGGTG VKVSCKASGYAFCCTCTGTCAAAGTGTCTTGCAAGGCAAGCGGCTACGCG TTTGMQWVRQATTTACGACCACCGGCATGCAGTGGGTCCGTCAGGCCCC PGQGLEWMGWIGGGCCAGGGTCTGGAATGGATGGGCTGGATCAACACCC NTHSGVPKYVEDATTCTGGCGTACCGAAATACGTTGAAGATTTCAAAGGC FKGRFVFSLDTSVCGTTTCGTGTTCTCCCTGGATACGTCCGTTTCCACCGCC STAYLQISSLKAETACCTGCAGATCTCTTCCCTGAAAGCAGAAGATACTGC DTAVYYCARSGNGGTGTACTATTGCGCACGTAGCGGCAACGGCAACTACG GNYDLAYFKYWACCTGGCCTACTTCAAATACTGGGGTCAGGGTACTCTG GQGTLVTVS GTGACCGTATCCTCT anti-ATGGAAGACATTCAGATGACCCAGAGCCCGTCCTCCCT 710 MEDIQMTQSPSSL 735 Her2-GAGCGCTTCTGTTGGCGACCGCGTGACCATCACCTGCC SASVGDRVTITCR AE864GTGCTTCCCAGGATGTTAACACCGCTGTAGCTTGGTATC ASQDVNTAVAWAACAGAAACCGGGCAAAGCACCGAAACTGCTGATCTA YQQKPGKAPKLLCTCTGCTTCCTTTCTGTATAGCGGTGTTCCGTCTCGTTTC IYSASFLYSGVPSAGCGGCTCTCGTAGCGGTACGGATTTTACTCTGACGAT RFSGSRSGTDFTLCAGCTCTCTGCAGCCGGAGGACTTCGCTACCTACTACT TISSLQPEDFATYGCCAGCAGCACTACACCACCCCGCCTACCTTTGGTCAG YCQQHYTTPPTFGGCACCAAAGTGGAAATCAAGACCGGTTCTGGCGAAG GQGTKVEIKTGSGCTCTGAAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGT GEGSEGEGGGEGGAAGGATCTGGTGAAGGTGGCGAAGGTGAGGGATCTG SEGEGSGEGGEGGTACCGAGGTCCAGCTGGTTGAGTCTGGCGGCGGTCTG EGSGTEVQLVESGTCCAACCTGGTGGCTCCCTGCGCCTGTCTTGCGCAGCG GGGLVQPGGSLRTCCGGCTTTAATATCAAAGATACGTACATTCACTGGGTC LSCAASGFNIKDTCGCCAGGCACCGGGCAAAGGCCTGGAATGGGTTGCTCG YIHWVRQAPGKGTATCTACCCGACTAACGGTTATACCCGTTATGCAGACA LEWVARIYPTNGGCGTAAAGGGTCGCTTCACGATCTCCGCGGATACCTCC YTRYADSVKGRFAAAAACACCGCATACCTGCAAATGAACTCTCTGCGTGC TISADTSKNTAYLGGAAGATACTGCCGTGTACTACTGCTCTCGCTGGGGCG QMNSLRAEDTAVGTGACGGTTTCTATGCAATGGACTACTGGGGTCAAGGT YYCSRWGGDGFACTCTGGTAACTGTTTCCGGGTCTCCAGGTAGCCCGGCT YAMDYWGQGTLGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAG VTVSGSPGSPAGSCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACC PTSTEEGTSESATGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTC PESGPGTSTEPSECGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCC GSAPGSPAGSPTSGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGA TEEGTSTEPSEGSGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGG APGTSTEPSEGSAAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCT PGTSESATPESGPGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGA GSEPATSGSETPGAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTG SEPATSGSETPGSAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGC PAGSPTSTEEGTSCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACC ESATPESGPGTSTAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAG EPSEGSAPGTSTEGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGT PSEGSAPGSPAGSACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAC PTSTEEGTSTEPSCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTC EGSAPGTSTEPSETGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTA GSAPGTSESATPECTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAA SGPGTSTEPSEGSAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGC APGTSESATPESGTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACC PGSEPATSGSETPGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGT GTSTEPSEGSAPGCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACC TSTEPSEGSAPGTCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCC SESATPESGPGTSGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCT ESATPESGPGSPACCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAA GSPTSTEEGTSESTCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGA ATPESGPGSEPATAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTG SGSETPGTSESATGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCT PESGPGTSTEPSECCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACC GSAPGTSTEPSEGAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAG SAPGTSTEPSEGSGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGT APGTSTEPSEGSAACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTAC PGTSTEPSEGSAPTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCC GTSTEPSEGSAPGCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCT SPAGSPTSTEEGTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGA STEPSEGSAPGTSAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTG ESATPESGPGSEPCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGC ATSGSETPGTSESGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAAC ATPESGPGSEPATCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTA SGSETPGTSESATCTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCG PESGPGTSTEPSEAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCT GSAPGTSESATPEGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTC SGPGSPAGSPTSTCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTA EEGSPAGSPTSTECTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACT EGSPAGSPTSTEEGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGG GTSESATPESGPGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCAC TSTEPSEGSAPGTCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCA SESATPESGPGSEGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGG PATSGSETPGTSETACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTA SATPESGPGSEPAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC TSGSETPGTSESATCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCT TPESGPGTSTEPSACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGC EGSAPGSPAGSPTTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAA STEEGTSESATPEGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCA SGPGSEPATSGSEACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGC TPGTSESATPESGTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTC PGSPAGSPTSTEECGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCA GSPAGSPTSTEEGACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCCGA TSTEPSEGSAPGTGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTG SESATPESGPGTSAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAA ESATPESGPGTSETCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATC SATPESGPGSEPATGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAA TSGSETPGSEPATCCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACT SGSETPGSPAGSPCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGA TSTEEGTSTEPSEAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAG GSAPGTSTEPSEGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGT SAPGSEPATSGSEAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTAC TPGTSESATPESGCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTC PGTSTEPSEGSAPTACTGAACCGTCCGAGGGCAGCGCACCA anti-ATGGAAGACATTCAGATGACCCAGAGCCCGTCCTCCCT 711 MEDIQMTQSPSSL 736 Her2-GAGCGCTTCTGTTGGCGACCGCGTGACCATCACCTGCC SASVGDRVTITCR AE576GTGCTTCCCAGGATGTTAACACCGCTGTAGCTTGGTATC ASQDVNTAVAWAACAGAAACCGGGCAAAGCACCGAAACTGCTGATCTA YQQKPGKAPKLLCTCTGCTTCCTTTCTGTATAGCGGTGTTCCGTCTCGTTTC IYSASFLYSGVPSAGCGGCTCTCGTAGCGGTACGGATTTTACTCTGACGAT RFSGSRSGTDFTLCAGCTCTCTGCAGCCGGAGGACTTCGCTACCTACTACT TISSLQPEDFATYGCCAGCAGCACTACACCACCCCGCCTACCTTTGGTCAG YCQQHYTTPPTFGGCACCAAAGTGGAAATCAAGACCGGTTCTGGCGAAG GQGTKVEIKTGSGCTCTGAAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGT GEGSEGEGGGEGGAAGGATCTGGTGAAGGTGGCGAAGGTGAGGGATCTG SEGEGSGEGGEGGTACCGAGGTCCAGCTGGTTGAGTCTGGCGGCGGTCTG EGSGTEVQLVESGTCCAACCTGGTGGCTCCCTGCGCCTGTCTTGCGCAGCG GGGLVQPGGSLRTCCGGCTTTAATATCAAAGATACGTACATTCACTGGGTC LSCAASGFNIKDTCGCCAGGCACCGGGCAAAGGCCTGGAATGGGTTGCTCG YIHWVRQAPGKGTATCTACCCGACTAACGGTTATACCCGTTATGCAGACA LEWVARIYPTNGGCGTAAAGGGTCGCTTCACGATCTCCGCGGATACCTCC YTRYADSVKGRFAAAAACACCGCATACCTGCAAATGAACTCTCTGCGTGC TISADTSKNTAYLGGAAGATACTGCCGTGTACTACTGCTCTCGCTGGGGCG QMNSLRAEDTAVGTGACGGTTTCTATGCAATGGACTACTGGGGTCAAGGT YYCSRWGGDGFACTCTGGTAACTGTTTCCGGGTCTCCAGGTAGCCCGGCT YAMDYWGQGTLGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAG VTVSGSPGSPAGSCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACC PTSTEEGTSESATGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTC PESGPGTSTEPSECGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCC GSAPGSPAGSPTSGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGA TEEGTSTEPSEGSGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGG APGTSTEPSEGSAAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCT PGTSESATPESGPGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGA GSEPATSGSETPGAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTG SEPATSGSETPGSAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGC PAGSPTSTEEGTSCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACC ESATPESGPGTSTAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAG EPSEGSAPGTSTEGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGT PSEGSAPGSPAGSACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAC PTSTEEGTSTEPSCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTC EGSAPGTSTEPSETGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTA GSAPGTSESATPECTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAA SGPGTSTEPSEGSAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGC APGTSESATPESGTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACC PGSEPATSGSETPGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGT GTSTEPSEGSAPGCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACC TSTEPSEGSAPGTCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCC SESATPESGPGTSGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCT ESATPESGPGSPACCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAA GSPTSTEEGTSESTCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGA ATPESGPGSEPATAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTG SGSETPGTSESATGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCT PESGPGTSTEPSECCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACC GSAPGTSTEPSEGAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAG SAPGTSTEPSEGSGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGT APGTSTEPSEGSAACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTAC PGTSTEPSEGSAPTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCC GTSTEPSEGSAPGCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCT SPAGSPTSTEEGTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGA STEPSEGSAPGTSAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTG ESATPESGPGSEPCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGC ATSGSETPGTSESGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAAC ATPESGPGSEPATCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTA SGSETPGTSESATCTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCG PESGPGTSTEPSEAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTCCT GSAPGTSESATPEGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTC SGPGSPAGSPTSTCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTA EEGSPAGSPTSTECTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACT EGSPAGSPTSTEEGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGG GTSESATPESGPGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCAC TSTEPSEGSAP CA anti-ATGGAAGACATTCAGATGACCCAGAGCCCGTCCTCCCT 712 MEDIQMTQSPSSL 737 Her2-GAGCGCTTCTGTTGGCGACCGCGTGACCATCACCTGCC SASVGDRVTITCR AE288GTGCTTCCCAGGATGTTAACACCGCTGTAGCTTGGTATC ASQDVNTAVAWAACAGAAACCGGGCAAAGCACCGAAACTGCTGATCTA YQQKPGKAPKLLCTCTGCTTCCTTTCTGTATAGCGGTGTTCCGTCTCGTTTC IYSASFLYSGVPSAGCGGCTCTCGTAGCGGTACGGATTTTACTCTGACGAT RFSGSRSGTDFTLCAGCTCTCTGCAGCCGGAGGACTTCGCTACCTACTACT TISSLQPEDFATYGCCAGCAGCACTACACCACCCCGCCTACCTTTGGTCAG YCQQHYTTPPTFGGCACCAAAGTGGAAATCAAGACCGGTTCTGGCGAAG GQGTKVEIKTGSGCTCTGAAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGT GEGSEGEGGGEGGAAGGATCTGGTGAAGGTGGCGAAGGTGAGGGATCTG SEGEGSGEGGEGGTACCGAGGTCCAGCTGGTTGAGTCTGGCGGCGGTCTG EGSGTEVQLVESGTCCAACCTGGTGGCTCCCTGCGCCTGTCTTGCGCAGCG GGGLVQPGGSLRTCCGGCTTTAATATCAAAGATACGTACATTCACTGGGTC LSCAASGFNIKDTCGCCAGGCACCGGGCAAAGGCCTGGAATGGGTTGCTCG YIHWVRQAPGKGTATCTACCCGACTAACGGTTATACCCGTTATGCAGACA LEWVARIYPTNGGCGTAAAGGGTCGCTTCACGATCTCCGCGGATACCTCC YTRYADSVKGRFAAAAACACCGCATACCTGCAAATGAACTCTCTGCGTGC TISADTSKNTAYLGGAAGATACTGCCGTGTACTACTGCTCTCGCTGGGGCG QMNSLRAEDTAVGTGACGGTTTCTATGCAATGGACTACTGGGGTCAAGGT YYCSRWGGDGFACTCTGGTAACTGTTTCCGGGTCTCCAGGTACCTCTGAA YAMDYWGQGTLAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGC VTVSGSPGTSESATACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCG TPESGPGSEPATSCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACC GSETPGTSESATPTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTAC ESGPGSEPATSGSTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGA ETPGTSESATPESGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCT GPGTSTEPSEGSACCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAA PGSPAGSPTSTEETCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGA GTSESATPESGPGAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCG SEPATSGSETPGTGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAG SESATPESGPGSPGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGA AGSPTSTEEGSPAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAG GSPTSTEEGTSTEGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGT PSEGSAPGTSESAACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTAC TPESGPGTSESATTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCG PESGPGTSESATPAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAA ESGPGSEPATSGSCCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGC ETPGSEPATSGSEAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTG TPGSPAGSPTSTEAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAA EGTSTEPSEGSAPCCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAAC GTSTEPSEGSAPGCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTA SEPATSGSETPGTCTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCG SESATPESGPGTS AGGGCAGCGCACCATEPSEGSAP AE912- ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGA 713MAEPAGSPTSTEE 738 anti- AGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGGTPGSGTASSSPG Her2 TAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGC SSTPSGATGSPGATTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCCC SPGTSSTGSPGSPGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGA AGSPTSTEEGTSEAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTG SATPESGPGTSTEAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGC PSEGSAPGSPAGSTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCT PTSTEEGTSTEPSTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTC EGSAPGTSTEPSETGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCC GSAPGTSESATPECGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGT SGPGSEPATSGSETCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTC TPGSEPATSGSETTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTA PGSPAGSPTSTEECTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCC GTSESATPESGPGGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGC TSTEPSEGSAPGTACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCAC STEPSEGSAPGSPCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAA AGSPTSTEEGTSTGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGG EPSEGSAPGTSTETACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA PSEGSAPGTSESACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACT TPESGPGTSTEPSTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTC EGSAPGTSESATPTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAAC ESGPGSEPATSGSCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCG ETPGTSTEPSEGSAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAA APGTSTEPSEGSACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGC PGTSESATPESGPAACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAA GTSESATPESGPGCCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCA SPAGSPTSTEEGTACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCC SESATPESGPGSETGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTT PATSGSETPGTSECTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAG SATPESGPGTSTETCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAG PSEGSAPGTSTEPCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG SEGSAPGTSTEPSCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCT EGSAPGTSTEPSECCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCC GSAPGTSTEPSEGAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAG SAPGTSTEPSEGSGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT APGSPAGSPTSTEAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTAC EGTSTEPSEGSAPTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCT GTSESATPESGPGCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAA SEPATSGSETPGTCCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAA SESATPESGPGSEAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGC PATSGSETPGTSEAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCG SATPESGPGTSTECTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGT PSEGSAPGTSESACCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACT TPESGPGSPAGSPCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGAC TSTEEGSPAGSPTTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTT STEEGSPAGSPTSCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCT TEEGTSESATPESACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTC GPGTSTEPSEGSACGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCG PGTSESATPESGPCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGC GSEPATSGSETPGCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCC TSESATPESGPGSAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAG EPATSGSETPGTSGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT ESATPESGPGTSTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTAC EPSEGSAPGSPAGTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCC SPTSTEEGTSESACTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCT TPESGPGSEPATSGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACC GSETPGTSESATPGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAA ESGPGSPAGSPTSGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGC TEEGSPAGSPTSTTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTC EEGTSTEPSEGSATCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTC PGTSESATPESGPCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCC GTSESATPESGPGCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCT TSESATPESGPGSGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGA EPATSGSETPGSEATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTG PATSGSETPGSPAAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAA GSPTSTEEGTSTEACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGA PSEGSAPGTSTEPGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCAC SEGSAPGSEPATSCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA GSETPGTSESATPGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGG ESGPGTSTEPSEGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTA SAPGSSSLDIQMTCTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTTCG QSPSSLSASVGDRTCTTCACTCGACATTCAGATGACCCAGAGCCCGTCCTCC VTITCRASQDVNCTGAGCGCTTCTGTTGGCGACCGCGTGACCATCACCTG TAVAWYQQKPGCCGTGCTTCCCAGGATGTTAACACCGCTGTAGCTTGGTA KAPKLLIYSASFLTCAACAGAAACCGGGCAAAGCACCGAAACTGCTGATCT YSGVPSRFSGSRSACTCTGCTTCCTTTCTGTATAGCGGTGTTCCGTCTCGTTT GTDFTLTISSLQPCAGCGGCTCTCGTAGCGGTACGGATTTTACTCTGACGA EDFATYYCQQHYTCAGCTCTCTGCAGCCGGAGGACTTCGCTACCTACTACT TTPPTFGQGTKVEGCCAGCAGCACTACACCACCCCGCCTACCTTTGGTCAG IKTGSGEGSEGEGGGCACCAAAGTGGAAATCAAGACCGGTTCTGGCGAAG GGEGSEGEGSGEGCTCTGAAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGT GGEGEGSGTEVQGAAGGATCTGGTGAAGGTGGCGAAGGTGAGGGATCTG LVESGGGLVQPGGTACCGAGGTCCAGCTGGTTGAGTCTGGCGGCGGTCTG GSLRLSCAASGFGTCCAACCTGGTGGCTCCCTGCGCCTGTCTTGCGCAGCG NIKDTYIHWVRQTCCGGCTTTAATATCAAAGATACGTACATTCACTGGGTC APGKGLEWVARICGCCAGGCACCGGGCAAAGGCCTGGAATGGGTTGCTCG YPTNGYTRYADSTATCTACCCGACTAACGGTTATACCCGTTATGCAGACA VKGRFTISADTSKGCGTAAAGGGTCGCTTCACGATCTCCGCGGATACCTCC NTAYLQMNSLRAAAAAACACCGCATACCTGCAAATGAACTCTCTGCGTGC EDTAVYYCSRWGGAAGATACTGCCGTGTACTACTGCTCTCGCTGGGGCG GGDGFYAMDYWGTGACGGTTTCTATGCAATGGACTACTGGGGTCAAGGT GQGTLVTVS ACTCTGGTAACTGTTTCCanti- ATGGAGGATATTCTGCTGACGCAAAGCCCTGTTATTCT 714 MEDTELTQSPVTE 739 EGFR-GTCTGTTAGCCCGGGTGAGCGCGTTAGCTTCAGCTGCC SVSPGERVSFSCR Y576-GTGCATCTCAGAGCATTGGCACGAACATTCATTGGTAT ASQSIGTNIHWY FLAG-CAACAACGTACCAACGGTAGCCCGCGTCTGCTGATTAA QQRTNGSPRLLIK HIS6ATACGCATCCGAATCTATCTCTGGTATCCCGTCTCGCTT YASESISGIPSRFS (“His6”CAGCGGTTCTGGTAGCGGCACCGACTTTACCCTGAGCA GSGSGTDFTLSIN disclosedTTAACTCTGTAGAAAGCGAAGATATTGCGGATTACTAC SVESEDIADYYC as SEQ IDTGCCAGCAGAACAACAACTGGCCGACTACTTTTGGTGC QQNNNWPTTFGA NO: 218)AGGTACTAAACTGGAACTGAAAACCGGTTCTGGCGAAG GTKLELKTGSGEGCTCTGAAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGT GSEGEGGGEGSEGAAGGATCTGGTGAAGGTGGCGAAGGTGAGGGATCTG GEGSGEGGEGEGGTACCCAAGTGCAGCTGAAACAGAGCGGTCCGGGTCTG SGTQVQLKQSGPGTGCAACCATCCCAGTCTCTGTCTATTACCTGTACCGTT GLVQPSQSLSITCAGCGGTTTCTCCCTGACCAACTACGGTGTTCACTGGGTT TVSGFSLTNYGVCGCCAGTCCCCAGGCAAAGGCCTGGAATGGCTGGGCGT HWVRQSPGKGLETATTTGGTCCGGCGGCAATACGGATTATAACACCCCGT WLGVIWSGGNTDTCACCTCTCGTCTGTCTATCAACAAAGATAATTCTAAAA YNTPFTSRLSINKGCCAGGTATTCTTCAAGATGAACTCTCTGCAGAGCAAT DNSKSQVFFKMNGACACCGCCATCTACTATTGCGCTCGTGCCCTGACTTAC SLQSNDTAIYYCTACGATTACGAGTTCGCATATTGGGGCCAGGGCACTCT ARALTYYDYEFAGGTGACCGTTTCCGGAGGTGAGGGTTCTGGCGAAGGTT YWGQGTLVTVSCCGAAGGTGAGGGCTCCGAAGGATCTGGCGAAGGTGA GGEGSGEGSEGEGGGTTCCGAAGGTTCTGGCGAAGGTGAAGGCGGTTCTG GSEGSGEGEGSEAGGGATCCGAAGGTGAAGGCTCCGAAGGATCTGGCGA GSGEGEGGSEGSAGGTGAAGGTGGTGAAGGTTCTGGCGAAGGTGAGGGA EGEGSEGSGEGETCTGGCGAAGGCTCTGAAGGTGAAGGTGGTGGTGAAGG GGEGSGEGEGSGCTCTGAAGGTGAAGGATCTGGTGAAGGTGGCGAAGGTG EGSEGEGGGEGSAGGGATCTGAAGGCGGCTCCGAAGGTGAAGGCGGATC EGEGSGEGGEGETGAAGGCGGCGAAGGTGAAGGTTCCGAAGGTTCTGGTG GSEGGSEGEGGSAAGGTGAAGGATCTGAAGGTGGCTCCGAAGGTGAAGG EGGEGEGSEGSGATCTGAAGGCGGTTCCGAAGGTGAGGGCTCTGAAGGTT EGEGSEGGSEGECTGGCGAAGGTGAAGGCTCTGAAGGATCTGGTGAAGGT GSEGGSEGEGSEGAAGGTTCCGAAGGTTCTGGTGAAGGTGAAGGTTCCGA GSGEGEGSEGSGAGGTTCTGGCGAAGGTGAAGGTTCTGAAGGTGGCTCTG EGEGSEGSGEGEAAGGTGAAGGCGGCTCTGAAGGATCCGAAGGTGAAGG GSEGSGEGEGSETTCTGGTGAAGGCTCTGAAGGTGAAGGCGGCTCTGAGG GGSEGEGGSEGSGTTCCGAAGGTGAAGGCGGAGGCGAAGGTTCTGAAGG EGEGSGEGSEGETGAGGGATCTGGTGAAGGTTCTGAAGGTGAAGGCGGTT GGSEGSEGEGGGCTGAAGGTTCCGAAGGTGAAGGTGGCTCTGAGGGATCC EGSEGEGSGEGSEGAAGGTGAAGGTGGCGAAGGATCTGGTGAAGGTGAAG GEGGSEGSEGEGGTTCTGAAGGTTCTGGCGAAGGTGAGGGTTCTGGCGAA GSEGSEGEGGEGGGTTCCGAAGGTGAGGGCTCCGAAGGATCTGGCGAAG SGEGEGSEGSGEGTGAGGGTTCCGAAGGTTCTGGCGAAGGTGAAGGCGGT GEGSGEGSEGEGTCTGAGGGATCCGAAGGTGAGGGTTCTGGCGAAGGTTC SEGSGEGEGSEGSCGAAGGTGAGGGCTCCGAAGGATCTGGCGAAGGTGAG GEGEGGSEGSEGGGTTCCGAAGGTTCTGGCGAAGGTGAAGGCGGTTCTGA EGSGEGSEGEGSEGGGATCCGAAGGTGAAGGCGGTTCTGAAGGTTCCGAAG GSGEGEGSEGSGGTGAAGGTGGCTCTGAGGGATCCGAAGGTGAAGGTGG EGEGGSEGSEGECGAAGGATCTGGTGAAGGTGAAGGTTCTGAAGGTTCTG GGSEGSEGEGGSGCGAAGGTGAGGGTTCTGGCGAAGGTTCCGAAGGTGA EGSEGEGGEGSGGGGCTCCGAAGGATCTGGCGAAGGTGAGGGTTCCGAA EGEGSEGSGEGEGGTTCTGGCGAAGGTGAAGGCGGTTCTGAGGGATCCGA GSGEGSEGEGSEAGGTGAAGGCTCCGAAGGATCTGGCGAAGGTGAAGGT GSGEGEGSEGSGGGTGAAGGTTCTGGCGAAGGTGAGGGATCTGGCGAAG EGEGGSEGSEGEGCTCTGAAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGT GSEGSGEGEGGEGAAGGTTCCGAAGGTTCTGGTGAAGGTGAAGGTTCCGA GSGEGEGSGEGSAGGTTCTGGCGAAGGTGAAGGTTCTGAAGGTGGCTCTG EGEGGGEGSEGEAAGGTGAAGGCGGCTCTGAAGGATCCGAAGGTGAAGG GSEGSGEGEGSEATCTGAAGGTGGCTCCGAAGGTGAAGGATCTGAAGGCG GSGEGEGSEGGSGTTCCGAAGGTGAGGGCTCTGAAGGTTCTGGCGAAGGT EGEGGSEGSEGEGAAGGCTCTGAAGGATCTGGTGAAGGTGAAGGATCTGG GSEGGSEGEGSECGAAGGCTCCGAAGGTGAAGGCGGTTCTGAAGGTGGC GGSEGEGSEGSGGAAGGTGAAGGATCTGAAGGTGGTTCCGAAGGTGAGG EGEGSEGSGEGEGATCTGAAGGTGGCTCTGAAGGTGAAGGTGGCGAAGGT GSGEGSEGEGGSTCTGGCGAAGGTGAAGGTGGAGGCGAAGGTTCTGAAG EGGEGEGSEGGSGTGAAGGTTCCGAAGGTTCTGGTGAAGGTGAGGGATCT EGEGSEGGSEGEGGCGAAGGTTCTGAAGGTGATTATAAAGACGATGACGA GGEGSGEGEGGGTAAAGGTGGTTCTCATCACCATCACCATCACTAA EGSEGEGSEGSGE GEGSGEGSEGDYKDDDDKGGSHH HHHH anti-CD3- ATGAAAGACATCCAGATGACCCAGTCTCCTTCCTCTCTG 715MKDIQMTQSPSS 740 Y288- TCCGCGTCCGTGGGCGACCGTGTTACTATCACCTGCTCCLSASVGDRVTITC GFP- GCCTCCTCTTCTGTCAGCTACATGAACTGGTATCAGCAG SASSSVSYMNWYHIS8 ACTCCTGGCAAAGCTCCAAAACGTTGGATTTACGATAC QQTPGKAPKRWI (“His8”GTCCAAGCTGGCCTCCGGCGTACCAAGCCGTTTCTCTG YDTSKLASGVPS disclosedGCTCTGGCAGCGGCACGGATTACACCTTCACTATTTCTA RFSGSGSGTDYTF as SEQ IDGCCTGCAGCCTGAAGATATTGCCACCTATTACTGCCAA TISSLQPEDIATY NO: 697)CAATGGTCCTCCAATCCTTTTACCTTTGGTCAGGGCACT YCQQWSSNPFTFAAGCTGCAGATTACTCGCACCGGTTCTGGCGAAGGCTC GQGTKLQITRTGSTGAAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGTGAA GEGSEGEGGGEGGGATCTGGTGAAGGTGGCGAAGGTGAGGGATCTGGTAC SEGEGSGEGGEGCCAGGTCCAACTGGTTCAATCCGGCGGCGGTGTAGTTC EGSGTQVQLVQSAACCGGGTCGCTCTCTGCGTCTTTCCTGCAAGGCGTCCG GGGVVQPGRSLRGTTACACTTTCACGCGTTACACCATGCACTGGGTCCGTC LSCKASGYTFTRAGGCTCCTGGTAAAGGTCTGGAATGGATTGGCTATATC YTMHWVRQAPGAACCCGTCTCGCGGCTATACCAACTATAACCAGAAATT KGLEWIGYINPSRCAAAGATCGTTTTACGATTTCCACTGATAAATCCAAAA GYTNYNQKFKDRGCACCGCATTCCTCCAAATGGACAGCCTGCGTCCGGAA FTISTDKSKSTAFGACACGGCGGTTTATTATTCCGCCCGTTACTACGATGAC LQMDSLRPEDTACACTACTGCCTGGATTATTGGGGCCAAGGCACTCCAGT VYYSARYYDDHAACCGTGAGCAGCGGAGGTGAGGGTTCTGGCGAAGGTT YCLDYWGQGTPCCGAAGGTGAGGGCTCCGAAGGATCTGGCGAAGGTGA VTVSSGGEGSGEGGGTTCCGAAGGTTCTGGCGAAGGTGAAGGCGGTTCTG GSEGEGSEGSGEAGGGATCCGAAGGTGAAGGCGGTTCTGAGGGATCTGA GEGSEGSGEGEGAGGTGAAGGTGGCTCTGAAGGATCTGAAGGTGAGGGA GSEGSEGEGGSETCTGGTGAAGGTTCTGAAGGTGAAGGCGGCTCTGAGGG GSEGEGGSEGSETTCTGAAGGTGAAGGATCTGGTGAAGGTTCCGAAGGTG GEGSGEGSEGEGAGGGTTCTGAAGGTGGTTCTGAAGGTGAAGGCGGTTCT GSEGSEGEGSGEGAGGGTTCTGAAGGTGAGGGTTCTGGCGAAGGTTCCGA GSEGEGSEGGSEAGGTGAAGGCGGCGAAGGTGGATCTGAAGGTGAGGGC GEGGSEGSEGEGTCCGAAGGATCTGGCGAAGGTGAAGGTTCTGGCGAAGG SGEGSEGEGGEGTTCCGAAGGTGAAGGTTCTGAAGGATCTGGCGAAGGTG GSEGEGSEGSGEAGGGTTCTGGCGAAGGTTCCGAAGGTGAGGGCTCCGAA GEGSGEGSEGEGGGATCTGGCGAAGGTGAGGGTTCCGAAGGTTCTGGCGA SEGSGEGEGSGEAGGTGAAGGCGGTTCTGAGGGATCCGAAGGTGAAGGC GSEGEGSEGSGETCCGAAGGATCTGGCGAAGGTGAAGGTGGTGAAGGTTC GEGSEGSGEGEGTGGCGAAGGTGAGGGATCTGGCGAAGGCTCTGAAGGT GSEGSEGEGSEGSGAAGGTGGTGGTGAAGGCTCTGAAGGTGAAGGATCTG GEGEGGEGSGEGGTGAAGGTGGCGAAGGTGAGGGATCTGAAGGCGGCTC EGSGEGSEGEGGCGAAGGTGAAGGCGGATCTGAAGGCGGCGAAGGTGAA GEGSEGEGSGEGGGTTCCGAAGGTTCTGGTGAAGGTGAAGGATCTGAAGG GEGEGSEGGSEGTGGCTCCGAAGGTGAAGGATCTGAAGGCGGTTCCGAAG EGGSEGGEGEGSGTGAGGGCTCTGAAGGTTCTGGCGAAGGTGAAGGCTCT EGSGEGEGSEGGGAAGGATCTGGTGAAGGTTCGTCTTCACTCGAGGGTAC SEGEGSEGGSEGECGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATT GSEGSGEGEGSEAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTG GSGEGSSSLEGTEGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTAC LFTGVVPILVELDCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCC GDVNGHKFSVSGATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCA EGEGDATYGKLTATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATG LKFICTTGKLPVPACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAG WPTLVTTFSYGVGAACGCACTATATCTTTCAAAGATGACGGGAACTACAA QCFSRYPDHMKRGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTG HDFFKSAMPEGYTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAA VQERTISFKDDGGATGGAAACATTCTCGGACACAAACTCGAGTACAACTA NYKTRAEVKFEGTAACTCACACAATGTATACATCACGGCAGACAAACAAA DTLVNRTELKGIDAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAAC FKEDGNILGHKLATTGAAGATGGATCCGTTCAACTAGCAGACCATTATCA EYNYNSHNVYITACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACC ADKQKNGIKANFAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGA KIRHNIEDGSVQLAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTT ADHYQQNTPIGDGAGTTTGTAACTGCTGCTGGGATTGGTGGCTCTCATCAC GPVLLPDNHYLSCATCACCATCACCATCACTAA TQSALSKDPNEK RDHMVLLEFVTA AGIGGSHHHHHH HH anti-ATGGAGGACATTCAGATGACCCAGAGCCCGTCCTCCCT 716 MEDIQMTQSPSSL 741 Her2-GAGCGCTTCTGTTGGCGACCGCGTGACCATCACCTGCC SASVGDRVTITCR Y288-GTGCTTCCCAGGATGTTAACACCGCTGTAGCTTGGTATC ASQDVNTAVAW anti-CD3-AACAGAAACCGGGCAAAGCACCGAAACTGCTGATCTA YQQKPGKAPKLL HA-His6,CTCTGCTTCCTTTCTGTATAGCGGTGTTCCGTCTCGTTTC IYSASFLYSGVPS AC48AGCGGCTCTCGTAGCGGTACGGATTTTACTCTGACGAT RFSGSRSGTDFTL (“His6”CAGCTCTCTGCAGCCGGAGGACTTCGCTACCTACTACT TISSLQPEDFATY disclosedGCCAGCAGCACTACACCACCCCGCCTACCTTTGGTCAG YCQQHYTTPPTF as SEQ IDGGCACCAAAGTGGAAATCAAGACCGGTTCTGGCGAAG GQGTKVEIKTGS NO: 218)GCTCTGAAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGT GEGSEGEGGGEGGAAGGATCTGGTGAAGGTGGCGAAGGTGAGGGATCTG SEGEGSGEGGEGGTACCGAGGTCCAGCTGGTTGAGTCTGGCGGCGGTCTG EGSGTEVQLVESGTCCAACCTGGTGGCTCCCTGCGCCTGTCTTGCGCAGCG GGGLVQPGGSLRTCCGGCTTTAATATCAAAGATACGTACATTCACTGGGTC LSCAASGFNIKDTCGCCAGGCACCGGGCAAAGGCCTGGAATGGGTTGCTCG YIHWVRQAPGKGTATCTACCCGACTAACGGTTATACCCGTTATGCAGACA LEWVARIYPTNGGCGTAAAGGGTCGCTTCACGATCTCCGCGGATACCTCC YTRYADSVKGRFAAAAACACCGCATACCTGCAAATGAACTCTCTGCGTGC TISADTSKNTAYLGGAAGATACTGCCGTGTACTACTGCTCTCGCTGGGGCG QMNSLRAEDTAVGTGACGGTTTCTATGCAATGGACTACTGGGGTCAAGGT YYCSRWGGDGFACTCTGGTAACTGTTTCCGGAGGTGAGGGTTCTGGCGA YAMDYWGQGTLAGGTTCCGAAGGTGAGGGCTCCGAAGGATCTGGCGAA VTVSGGEGSGEGGGTGAGGGTTCCGAAGGTTCTGGCGAAGGTGAAGGCG SEGEGSEGSGEGEGTTCTGAGGGATCCGAAGGTGAAGGCGGTTCTGAGGGA GSEGSGEGEGGSTCTGAAGGTGAAGGTGGCTCTGAAGGATCTGAAGGTGA EGSEGEGGSEGSEGGGATCTGGTGAAGGTTCTGAAGGTGAAGGCGGCTCTG GEGGSEGSEGEGAGGGTTCTGAAGGTGAAGGATCTGGTGAAGGTTCCGAA SGEGSEGEGGSEGGTGAGGGTTCTGAAGGTGGTTCTGAAGGTGAAGGCGG GSEGEGSGEGSETTCTGAGGGTTCTGAAGGTGAGGGTTCTGGCGAAGGTT GEGSEGGSEGEGCCGAAGGTGAAGGCGGCGAAGGTGGATCTGAAGGTGA GSEGSEGEGSGEGGGCTCCGAAGGATCTGGCGAAGGTGAAGGTTCTGGCG GSEGEGGEGGSEAAGGTTCCGAAGGTGAAGGTTCTGAAGGATCTGGCGAA GEGSEGSGEGEGGGTGAGGGTTCTGGCGAAGGTTCCGAAGGTGAGGGCTC SGEGSEGEGSEGSCGAAGGATCTGGCGAAGGTGAGGGTTCCGAAGGTTCTG GEGEGSGEGSEGGCGAAGGTGAAGGCGGTTCTGAGGGATCCGAAGGTGA EGSEGSGEGEGSEAGGCTCCGAAGGATCTGGCGAAGGTGAAGGTGGTGAA GSGEGEGGSEGSGGTTCTGGCGAAGGTGAGGGATCTGGCGAAGGCTCTGA EGEGSEGSGEGEAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGTGAAGGA GGEGSGEGEGSGTCTGGTGAAGGTGGCGAAGGTGAGGGATCTGAAGGCG EGSEGEGGGEGSGCTCCGAAGGTGAAGGCGGATCTGAAGGCGGCGAAGG EGEGSGEGGEGETGAAGGTTCCGAAGGTTCTGGTGAAGGTGAAGGATCTG GSEGGSEGEGGSAAGGTGGCTCCGAAGGTGAAGGATCTGAAGGCGGTTCC EGGEGEGSEGSGGAAGGTGAGGGCTCTGAAGGTTCTGGCGAAGGTGAAG EGEGSEGGSEGEGCTCTGAAGGATCTGGTGAAGGTGACATCCAGATGACC GSEGGSEGEGSECAGTCTCCTTCCTCTCTGTCCGCGTCCGTGGGCGACCGT GSGEGEGSEGSGGTTACTATCACCTGCTCCGCCTCCTCTTCTGTCAGCTAC EGDIQMTQSPSSLATGAACTGGTATCAGCAGACTCCTGGCAAAGCTCCAAA SASVGDRVTITCSACGTTGGATTTACGATACGTCCAAGCTGGCCTCCGGCG ASSSVSYMNWYTACCAAGCCGTTTCTCTGGCTCTGGCAGCGGCACGGAT QQTPGKAPKRWITACACCTTCACTATTTCTAGCCTGCAGCCTGAAGATATT YDTSKLASGVPSGCCACCTATTACTGCCAACAATGGTCCTCCAATCCTTTT RFSGSGSGTDYTFACCTTTGGTCAGGGCACTAAGCTGCAGATTACTCGCAC TISSLQPEDIATYCGGTTCTGGCGAAGGCTCTGAAGGTGAAGGTGGTGGTG YCQQWSSNPFTFAAGGCTCTGAAGGTGAAGGATCTGGTGAAGGTGGCGA GQGTKLQITRTGSAGGTGAGGGATCTGGTACCCAGGTCCAACTGGTTCAAT GEGSEGEGGGEGCCGGCGGCGGTGTAGTTCAACCGGGTCGCTCTCTGCGT SEGEGSGEGGEGCTTTCCTGCAAGGCGTCCGGTTACACTTTCACGCGTTAC EGSGTQVQLVQSACCATGCACTGGGTCCGTCAGGCTCCTGGTAAAGGTCT GGGVVQPGRSLRGGAATGGATTGGCTATATCAACCCGTCTCGCGGCTATA LSCKASGYTFTRCCAACTATAACCAGAAATTCAAAGATCGTTTTACGATT YTMHWVRQAPGTCCACTGATAAATCCAAAAGCACCGCATTCCTCCAAAT KGLEWIGYINPSRGGACAGCCTGCGTCCGGAAGACACGGCGGTTTATTATT GYTNYNQKFKDRCCGCCCGTTACTACGATGACCACTACTGCCTGGATTATT FTISTDKSKSTAFGGGGCCAAGGCACTCCAGTAACCGTGAGCAGCGGCGG LQMDSLRPEDTATTATCCTTATGATGTTCCAGACTATGCAGGTGGCTCTCA VYYSARYYDDH TCACCATCACCATCACTGAYCLDYWGQGTP VTVSSGGYPYDV PDYAGGSHHHHH H anti-ATGGAGGACATTCAGATGACCCAGAGCCCGTCCTCCCT 717 MEDIQMTQSPSSL 742 Her2-GAGCGCTTCTGTTGGCGACCGCGTGACCATCACCTGCC SASVGDRVTITCR Y288-GTGCTTCCCAGGATGTTAACACCGCTGTAGCTTGGTATC ASQDVNTAVAW anti-AACAGAAACCGGGCAAAGCACCGAAACTGCTGATCTA YQQKPGKAPKLL EGFR-CTCTGCTTCCTTTCTGTATAGCGGTGTTCCGTCTCGTTTC IYSASFLYSGVPS HA-His6,AGCGGCTCTCGTAGCGGTACGGATTTTACTCTGACGAT RFSGSRSGTDFTL AC49CAGCTCTCTGCAGCCGGAGGACTTCGCTACCTACTACT TISSLQPEDFATY (“His6”GCCAGCAGCACTACACCACCCCGCCTACCTTTGGTCAG YCQQHYTTPPTF disclosedGGCACCAAAGTGGAAATCAAGACCGGTTCTGGCGAAG GQGTKVEIKTGS as SEQ IDGCTCTGAAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGT GEGSEGEGGGEG NO: 218)GAAGGATCTGGTGAAGGTGGCGAAGGTGAGGGATCTG SEGEGSGEGGEGGTACCGAGGTCCAGCTGGTTGAGTCTGGCGGCGGTCTG EGSGTEVQLVESGTCCAACCTGGTGGCTCCCTGCGCCTGTCTTGCGCAGCG GGGLVQPGGSLRTCCGGCTTTAATATCAAAGATACGTACATTCACTGGGTC LSCAASGFNIKDTCGCCAGGCACCGGGCAAAGGCCTGGAATGGGTTGCTCG YIHWVRQAPGKGTATCTACCCGACTAACGGTTATACCCGTTATGCAGACA LEWVARIYPTNGGCGTAAAGGGTCGCTTCACGATCTCCGCGGATACCTCC YTRYADSVKGRFAAAAACACCGCATACCTGCAAATGAACTCTCTGCGTGC TISADTSKNTAYLGGAAGATACTGCCGTGTACTACTGCTCTCGCTGGGGCG QMNSLRAEDTAVGTGACGGTTTCTATGCAATGGACTACTGGGGTCAAGGT YYCSRWGGDGFACTCTGGTAACTGTTTCCGGAGGTGAGGGTTCTGGCGA YAMDYWGQGTLAGGTTCCGAAGGTGAGGGCTCCGAAGGATCTGGCGAA VTVSGGEGSGEGGGTGAGGGTTCCGAAGGTTCTGGCGAAGGTGAAGGCG SEGEGSEGSGEGEGTTCTGAGGGATCCGAAGGTGAAGGCGGTTCTGAGGGA GSEGSGEGEGGSTCTGAAGGTGAAGGTGGCTCTGAAGGATCTGAAGGTGA EGSEGEGGSEGSEGGGATCTGGTGAAGGTTCTGAAGGTGAAGGCGGCTCTG GEGGSEGSEGEGAGGGTTCTGAAGGTGAAGGATCTGGTGAAGGTTCCGAA SGEGSEGEGGSEGGTGAGGGTTCTGAAGGTGGTTCTGAAGGTGAAGGCGG GSEGEGSGEGSETTCTGAGGGTTCTGAAGGTGAGGGTTCTGGCGAAGGTT GEGSEGGSEGEGCCGAAGGTGAAGGCGGCGAAGGTGGATCTGAAGGTGA GSEGSEGEGSGEGGGCTCCGAAGGATCTGGCGAAGGTGAAGGTTCTGGCG GSEGEGGEGGSEAAGGTTCCGAAGGTGAAGGTTCTGAAGGATCTGGCGAA GEGSEGSGEGEGGGTGAGGGTTCTGGCGAAGGTTCCGAAGGTGAGGGCTC SGEGSEGEGSEGSCGAAGGATCTGGCGAAGGTGAGGGTTCCGAAGGTTCTG GEGEGSGEGSEGGCGAAGGTGAAGGCGGTTCTGAGGGATCCGAAGGTGA EGSEGSGEGEGSEAGGCTCCGAAGGATCTGGCGAAGGTGAAGGTGGTGAA GSGEGEGGSEGSGGTTCTGGCGAAGGTGAGGGATCTGGCGAAGGCTCTGA EGEGSEGSGEGEAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGTGAAGGA GGEGSGEGEGSGTCTGGTGAAGGTGGCGAAGGTGAGGGATCTGAAGGCG EGSEGEGGGEGSGCTCCGAAGGTGAAGGCGGATCTGAAGGCGGCGAAGG EGEGSGEGGEGETGAAGGTTCCGAAGGTTCTGGTGAAGGTGAAGGATCTG GSEGGSEGEGGSAAGGTGGCTCCGAAGGTGAAGGATCTGAAGGCGGTTCC EGGEGEGSEGSGGAAGGTGAGGGCTCTGAAGGTTCTGGCGAAGGTGAAG EGEGSEGGSEGEGCTCTGAAGGATCTGGTGAAGGTGAGGATATTCTGCTG GSEGGSEGEGSEACGCAAAGCCCTGTTATTCTGTCTGTTAGCCCGGGTGA GSGEGEGSEGSGGCGCGTTAGCTTCAGCTGCCGTGCATCTCAGAGCATTG EGEDTELTQSPVTEGCACGAACATTCATTGGTATCAACAACGTACCAACGGT SVSPGERVSFSCRAGCCCGCGTCTGCTGATTAAATACGCATCCGAATCTAT ASQSIGTNIHWYCTCTGGTATCCCGTCTCGCTTCAGCGGTTCTGGTAGCGG QQRTNGSPRLLIKCACCGACTTTACCCTGAGCATTAACTCTGTAGAAAGCG YASESISGIPSRFSAAGATATTGCGGATTACTACTGCCAGCAGAACAACAAC GSGSGTDFTLSINTGGCCGACTACTTTTGGTGCAGGTACTAAACTGGAACT SVESEDIADYYCGAAAACCGGTTCTGGCGAAGGCTCTGAAGGTGAAGGTG QQNNNWPTTFGAGTGGTGAAGGCTCTGAAGGTGAAGGATCTGGTGAAGGT GTKLELKTGSGEGGCGAAGGTGAGGGATCTGGTACCCAAGTGCAGCTGA GSEGEGGGEGSEAACAGAGCGGTCCGGGTCTGGTGCAACCATCCCAGTCT GEGSGEGGEGEGCTGTCTATTACCTGTACCGTTAGCGGTTTCTCCCTGACC SGTQVQLKQSGPAACTACGGTGTTCACTGGGTTCGCCAGTCCCCAGGCAA GLVQPSQSLSITCAGGCCTGGAATGGCTGGGCGTTATTTGGTCCGGCGGCA TVSGFSLTNYGVATACGGATTATAACACCCCGTTCACCTCTCGTCTGTCTA HWVRQSPGKGLETCAACAAAGATAATTCTAAAAGCCAGGTATTCTTCAAG WLGVIWSGGNTDATGAACTCTCTGCAGAGCAATGACACCGCCATCTACTA YNTPFTSRLSINKTTGCGCTCGTGCCCTGACTTACTACGATTACGAGTTCGC DNSKSQVFFKMNATATTGGGGCCAGGGCACTCTGGTGACCGTTTCCGGCG SLQSNDTAIYYCGTTATCCTTATGATGTTCCAGACTATGCAGGTGGCTCTC ARALTYYDYEFAATCACCATCACCATCACTGA YWGQGTLVTVS GGYPYDVPDYAG GSHHHHHH anti-ATGGAGGACATTCAGATGACCCAGAGCCCGTCCTCCCT 718 MEDIQMTQSPSSL 743 Her2-GAGCGCTTCTGTTGGCGACCGCGTGACCATCACCTGCC SASVGDRVTITCR Y288-GTGCTTCCCAGGATGTTAACACCGCTGTAGCTTGGTATC ASQDVNTAVAW anti-CD3-AACAGAAACCGGGCAAAGCACCGAAACTGCTGATCTA YQQKPGKAPKLL HA-His8,CTCTGCTTCCTTTCTGTATAGCGGTGTTCCGTCTCGTTTC IYSASFLYSGVPS AC69AGCGGCTCTCGTAGCGGTACGGATTTTACTCTGACGAT RFSGSRSGTDFTL (“His8”CAGCTCTCTGCAGCCGGAGGACTTCGCTACCTACTACT TISSLQPEDFATY disclosedGCCAGCAGCACTACACCACCCCGCCTACCTTTGGTCAG YCQQHYTTPPTF as SEQ IDGGCACCAAAGTGGAAATCAAGACCGGTTCTGGCGAAG GQGTKVEIKTGS NO: 697)GCTCTGAAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGT GEGSEGEGGGEGGAAGGATCTGGTGAAGGTGGCGAAGGTGAGGGATCTG SEGEGSGEGGEGGTACCGAGGTCCAGCTGGTTGAGTCTGGCGGCGGTCTG EGSGTEVQLVESGTCCAACCTGGTGGCTCCCTGCGCCTGTCTTGCGCAGCG GGGLVQPGGSLRTCCGGCTTTAATATCAAAGATACGTACATTCACTGGGTC LSCAASGFNIKDTCGCCAGGCACCGGGCAAAGGCCTGGAATGGGTTGCTCG YIHWVRQAPGKGTATCTACCCGACTAACGGTTATACCCGTTATGCAGACA LEWVARIYPTNGGCGTAAAGGGTCGCTTCACGATCTCCGCGGATACCTCC YTRYADSVKGRFAAAAACACCGCATACCTGCAAATGAACTCTCTGCGTGC TISADTSKNTAYLGGAAGATACTGCCGTGTACTACTGCTCTCGCTGGGGCG QMNSLRAEDTAVGTGACGGTTTCTATGCAATGGACTACTGGGGTCAAGGT YYCSRWGGDGFACTCTGGTAACTGTTTCCGGGTCTCCAGGTGAGGGTTCT YAMDYWGQGTLGGCGAAGGTTCCGAAGGTGAGGGCTCCGAAGGATCTG VTVSGSPGEGSGGCGAAGGTGAGGGTTCCGAAGGTTCTGGCGAAGGTGA EGSEGEGSEGSGEAGGCGGTTCTGAGGGATCCGAAGGTGAAGGCGGTTCTG GEGSEGSGEGEGAGGGATCTGAAGGTGAAGGTGGCTCTGAAGGATCTGAA GSEGSEGEGGSEGGTGAGGGATCTGGTGAAGGTTCTGAAGGTGAAGGCG GSEGEGGSEGSEGCTCTGAGGGTTCTGAAGGTGAAGGATCTGGTGAAGGT GEGSGEGSEGEGTCCGAAGGTGAGGGTTCTGAAGGTGGTTCTGAAGGTGA GSEGSEGEGSGEAGGCGGTTCTGAGGGTTCTGAAGGTGAGGGTTCTGGCG GSEGEGSEGGSEAAGGTTCCGAAGGTGAAGGCGGCGAAGGTGGATCTGA GEGGSEGSEGEGAGGTGAGGGCTCCGAAGGATCTGGCGAAGGTGAAGGT SGEGSEGEGGEGTCTGGCGAAGGTTCCGAAGGTGAAGGTTCTGAAGGATC GSEGEGSEGSGETGGCGAAGGTGAGGGTTCTGGCGAAGGTTCCGAAGGTG GEGSGEGSEGEGAGGGCTCCGAAGGATCTGGCGAAGGTGAGGGTTCCGA SEGSGEGEGSGEAGGTTCTGGCGAAGGTGAAGGCGGTTCTGAGGGATCCG GSEGEGSEGSGEAAGGTGAAGGCTCCGAAGGATCTGGCGAAGGTGAAGG GEGSEGSGEGEGTGGTGAAGGTTCTGGCGAAGGTGAGGGATCTGGCGAAG GSEGSEGEGSEGSGCTCTGAAGGTGAAGGTGGTGGTGAAGGCTCTGAAGGT GEGEGGEGSGEGGAAGGATCTGGTGAAGGTGGCGAAGGTGAGGGATCTG EGSGEGSEGEGGAAGGCGGCTCCGAAGGTGAAGGCGGATCTGAAGGCGG GEGSEGEGSGEGCGAAGGTGAAGGTTCCGAAGGTTCTGGTGAAGGTGAAG GEGEGSEGGSEGGATCTGAAGGTGGCTCCGAAGGTGAAGGATCTGAAGGC EGGSEGGEGEGSGGTTCCGAAGGTGAGGGCTCTGAAGGTTCTGGCGAAGG EGSGEGEGSEGGTGAAGGCTCTGAAGGATCTGGTGAAGGTTCGTCTTCAC SEGEGSEGGSEGETCGAGGGTACCAAAGACATCCAGATGACCCAGTCTCCT GSEGSGEGEGSETCCTCTCTGTCCGCGTCCGTGGGCGACCGTGTTACTATC GSGEGSSSLEGTKACCTGCTCCGCCTCCTCTTCTGTCAGCTACATGAACTGG DIQMTQSPSSLSATATCAGCAGACTCCTGGCAAAGCTCCAAAACGTTGGAT SVGDRVTITCSASTTACGATACGTCCAAGCTGGCCTCCGGCGTACCAAGCC SSVSYMNWYQQGTTTCTCTGGCTCTGGCAGCGGCACGGATTACACCTTCA TPGKAPKRWIYDCTATTTCTAGCCTGCAGCCTGAAGATATTGCCACCTATT TSKLASGVPSRFSACTGCCAACAATGGTCCTCCAATCCTTTTACCTTTGGTC GSGSGTDYTFTISAGGGCACTAAGCTGCAGATTACTCGCACCGGTTCTGGC SLQPEDIATYYCQGAAGGCTCTGAAGGTGAAGGTGGTGGTGAAGGCTCTGA QWSSNPFTFGQGAGGTGAAGGATCTGGTGAAGGTGGCGAAGGTGAGGGA TKLQITRTGSGEGTCTGGTACCCAGGTCCAACTGGTTCAATCCGGCGGCGG SEGEGGGEGSEGTGTAGTTCAACCGGGTCGCTCTCTGCGTCTTTCCTGCAA EGSGEGGEGEGSGGCGTCCGGTTACACTTTCACGCGTTACACCATGCACTG GTQVQLVQSGGGGGTCCGTCAGGCTCCTGGTAAAGGTCTGGAATGGATTG VVQPGRSLRLSCGCTATATCAACCCGTCTCGCGGCTATACCAACTATAAC KASGYTFTRYTMCAGAAATTCAAAGATCGTTTTACGATTTCCACTGATAA HWVRQAPGKGLATCCAAAAGCACCGCATTCCTCCAAATGGACAGCCTGC EWIGYINPSRGYTGTCCGGAAGACACGGCGGTTTATTATTCCGCCCGTTACT NYNQKFKDRFTISACGATGACCACTACTGCCTGGATTATTGGGGCCAAGGC TDKSKSTAFLQMACTCCAGTAACCGTGAGCAGCACTAGTGGCGGTTATCC DSLRPEDTAVYYTTATGATGTTCCAGACTATGCAGGTGGCTCTCATCACCA SARYYDDHYCLD TCACCATCACCACCATTGAYWGQGTPVTVSS TSGGYPYDVPDY AGGSHHHHHHH H EGFR_VATGAAAGGGTCTCCAGGTGAAGTACAGCTTCAAGAATC 719 MKGSPGEVQLQE 744 HH1-TGGTGGTGGTCTTGTCCAGGCGGGCGATTCCCTGCGCCT SGGGLVQAGDSL AM144-GTCTTGTCTGGTCTCTGGTCGTTCATTTAACAGCTATAC RLSCLVSGRSFNS GFP6~22CATGGGCTGGTTCCGCCAAGCACCGGGCAAGGAACGTG YTMGWFRQAPG 9-H8,AATTCGTAGCAGCTATTCTCTGGTCCGGTCCTACGACCT KEREFVAAILWS LMS109.ACTATGCTGACTCTGTAAAAGGTCGCTTCACCATCTCCC GPTTYYADSVKG 005GTGATAACGCCAAAAACACCGTATATCTTCAGATGAAC RFTISRDNAKNTVTCTCTGAAACCGGAGGACACGGCCGTGTACTATTGTGC YLQMNSLKPEDTCGCTGCGCTGGGTGTACTGGTGCTAGCGCCTGGTAATG AVYYCAAALGVTCTACAGCTATTGGGGTCAAGGTACCCAGGTCACGGTA LVLAPGNVYSYWAGCTCCGCGCATCATGGAGGTACCCCGGGCAGCGGTAC GQGTQVTVSSAHCGCATCTTCCTCTCCAGGTAGCTCTACCCCGTCTGGTGC HGGTPGSGTASSSTACCGGTTCCCCAGGTAGCTCTACCCCGTCTGGTGCAAC PGSSTPSGATGSPCGGCTCCCCAGGTAGCCCGGCTGGCTCTCCTACCTCTAC GSSTPSGATGSPGTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTG SPAGSPTSTEEGTGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCT SESATPESGPGTSCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCA TEPSEGSAPGSSPGGTTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGT SASTGTGPGSSPSGCTTCTCCGGGTACTAGCTCTACTGGTTCTCCAGGTACC ASTGTGPGASPGTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTC TSSTGSPGTSTEPTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAAC SEGSAPGTSTEPSCGGCAACCTCCGGTTCTGAAACTCCAGGTTCGTCTTCAC EGSAPGSEPATSGTCGAGGGTACCGAACTTTTCACTGGAGTTGTCCCAATTC SETPGSSSLEGTETTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTT LFTGVVPILVELDCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGG GDVNGHKFSVSGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACT EGEGDATYGKLTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTA LKFICTTGKLPVPTGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAA WPTLVTTFSYGVACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTT QCFSRYPDHMKRATGTACAGGAACGCACTATATCTTTCAAAGATGACGGG HDFFKSAMPEGYAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGA VQERTISFKDDGTACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTT NYKTRAEVKFEGTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGT DTLVNRIELKGIDACAACTATAACTCACACAATGTATACATCACGGCAGAC FKEDGNILGHKLAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCG EYNYNSHNVYITCCACAACATTGAAGATGGATCCGTTCAACTAGCAGACC ADKQKNGIKANFATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTC KIRHNIEDGSVQLCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCC ADHYQQNTPIGDCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGT GPVLLPDNHYLSCCTTCTTGAGTTTGTAACTGCTGCTGGGATTGGTGGCTC TQSALSKDPNEKTCATCACCATCACCATCACCATCACTAA RDHMVLLEFVTA AGIGGSHHHHHH HH EGFR_VATGAAAGGGTCTCCAGGTGAAGTGCAGCTTCAAgAATC 720 MKGSPGEVQLQE 745 HH1-TGGTGGTGGTCTGGTACAAGCCGGTGATTCTCTGCGCCT SGGGLVQAGDSL AM144-GTCTTGTCTGGTCTCCGGTCGCTCTTTTAACAGCTATAC RLSCLVSGRSFNS GFP6~22CATGGGCTGGTTCCGCCAGGCACCAGGCAAAGAGCGTG YTMGWFRQAPG 9-H8,AATTCGTAGCAGCTATCCTGTGGTCTGGTCCGACTACCT KEREFVAAILWS LMS109.ACTATGCTGACTCTGTAAAGGGTCGCTTCACGATTTCCC GPTTYYADSVKG 020GTGATAACGCCAAAAACACGGTGTATCTACAAATGAAT RFTISRDNAKNTVTCTCTGAAACCGGAGGACACTGCCGTTTACTATTGTGCC YLQMNSLKPEDTGCTGCGCTGGGTGTACTGGTGCTTGCCCCTGGTAATGTA AVYYCAAALGVTACAGCTATTGGGGTCAAGGTACGCAAGTTACCGTGAG LVLAPGNVYSYWCTCTGCGCATCATGGAGGTACTTCTACCGAACCGTCCG GQGTQVTVSSAHAGGGCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAA HGGTSTEPSEGSAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCCGAAGG PGTSTEPSEGSAPTAGCGCACCAGGTTCTACCAGCGAATCCCCTTCTGGTA GTSTEPSEGSAPGCTGCTCCAGGTTCTACCAGCGAATCCCCTTCTGGCACCG STSESPSGTAPGSCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTC TSESPSGTAPGTSCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCA TPESGSASPGSEPGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGG ATSGSETPGTSESTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTA ATPESGPGTSTEPCTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACT SEGSAPGTSTEPSTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTC EGSAPGTSESATPTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTTCGTCTT ESGPGTSESATPECACTCGAGGGTACCGAACTTTTCACTGGAGTTGTCCCA SGPGSSSLEGTELATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAA FTGVVPILVELDGATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACAT DVNGHKFSVSGEACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGA GEGDATYGKLTLAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTC KFICTTGKLPVPWTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCAC PTLVTTFSYGVQATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGA CFSRYPDHMKRHAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATG DFFKSAMPEGYVACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAA QERTISFKDDGNGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATT YKTRAEVKFEGDGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACT TLVNRIELKGIDFCGAGTACAACTATAACTCACACAATGTATACATCACGG KEDGNILGHKLECAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAA YNYNSHNVYITAAATTCGCCACAACATTGAAGATGGATCCGTTCAACTAG DKQKNGIKANFKCAGACCATTATCAACAAAATACTCCAATTGGCGATGGC IRHNIEDGSVQLACCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAA DHYQQNTPIGDGTCTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCA PVLLPDNHYLSTCATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTGG QSALSKDPNEKRTGGCTCTCATCACCATCACCATCACCATCACTAA DHMVLLEFVTAA GIGGSHHHHHHH H EGFR_VATGAAAGGGTCTCCAGGTGAGGTTCAACTTCaAgAATCT 721 MKGSPGEVQLQE 746 HH1-GGTGGTGGTCTAGTACAAGCCGGCGACTCCCTGCGCCT SGGGLVQAGDSL AM144-GTCTTGTCTGGTCTCCGGTCGTTCTTTTAACAGCTATAC RLSCLVSGRSFNS GFP6~22CATGGGCTGGTTCCGCCAAGCTCCGGGCAAAGAACGTG YTMGWFRQAPG 9-H8,AATTCGTAGCAGCTATTCTCTGGTCTGGTCCTACCACCT KEREFVAAILWS LMS109.ACTATGCTGACTCTGTAAAGGGCCGTTTCACTATCTCCC GPTTYYADSVKG 038GTGATAACGCCAAAAACACTGTCTATCTGCAGATGAAT RFTISRDNAKNTVTCTCTGAAACCGGAGGACACCGCAGTATACTATTGCGC YLQMNSLKPEDTAGCTGCGCTGGGTGTACTGGTGCTCGCTCCAGGTAATG AVYYCAAALGVTATACAGCTATTGGGGTCAAGGTACGCAAGTCACGGTA LVLAPGNVYSYWAGCTCTGCGCATCATGGAGGTACCCCGGGCAGCGGTAC GQGTQVTVSSAHCGCATCTTCCTCTCCAGGTAGCTCTACCCCGTCTGGTGC HGGTPGSGTASSSTACCGGTTCCCCAGGTAGCTCTACCCCGTCTGGTGCAAC PGSSTPSGATGSPCGGCTCCCCAGGTAGCCCGGCTGGCTCTCCTACCTCTAC GSSTPSGATGSPGTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTG SPAGSPTSTEEGTGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCT SESATPESGPGTSCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCA TEPSEGSAPGSSPGGTTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGT SASTGTGPGSSPSGCTTCTCCGGGTACTAGCTCTACTGGTTCTCCAGGTACC ASTGTGPGASPGTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTC TSSTGSPGTSTEPTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAAC SEGSAPGTSTEPSCGGCAACCTCCGGTTCTGAAACTCCAGGTTCGTCTTCAC EGSAPGSEPATSGTCGAGGGTACCGAACTTTTCACTGGAGTTGTCCCAATTC SETPGSSSLEGTETTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTT LFTGVVPTEVELDCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGG GDVNGHKFSVSGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACT EGEGDATYGKLTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTA LKFICTTGKLPVPTGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAA WPTLVTTFSYGVACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTT QCFSRYPDHMKRATGTACAGGAACGCACTATATCTTTCAAAGATGACGGG HDFFKSAMPEGYAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGA VQERTISFKDDGTACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTT NYKTRAEVKFEGTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGT DTLVNRIELKGIDACAACTATAACTCACACAATGTATACATCACGGCAGAC FKEDGNILGHKLAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCG EYNYNSHNVYITCCACAACATTGAAGATGGATCCGTTCAACTAGCAGACC ADKQKNGIKANFATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTC KIRHNIEDGSVQLCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCC ADHYQQNTPIGDCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGT GPVLLPDNHYLSCCTTCTTGAGTTTGTAACTGCTGCTGGGATTGGTGGCTC TQSALSKDPNEKTCATCACCATCACCATCACCATCACTAA RDHMVLLEFVTA AGIGGSHHHHHH HH EGFR_VATGAAAGGGTCTCCAGGTGAAGTGCAACttcaAgAATCTG 722 MKGSPGEVQLQE 747 HH1-GTGGTGGTCTGGTACAAGCTGGTGACTCTCTGCGCCTGT SGGGLVQAGDSL AM144-CTTGTCTGGTCTCCGGTCGTTCCTTCAATAGCTATACCA RLSCLVSGRSFNS GFP6~22TGGGCTGGTTCCGCCAAGCGCCTGGCAAAGAGCGTGAA YTMGWFRQAPG 9-H8,TTCGTAGCAGCAATCCTTTGGTCCGGTCCAACTACCTAC KEREFVAAILWS LMS109.TATGCTGACTCTGTAAAAGGTCGCTTCACCATCTCCCGT GPTTYYADSVKG 045GATAACGCCAAAAACACTGTTTATCTACAAATGAATTC RFTISRDNAKNTVTCTGAAACCGGAGGACACGGCTGTTTACTACTGTGCTG YLQMNSLKPEDTCCGCGCTGGGTGTACTGGTGCTCGCACCAGGTAATGTG AVYYCAAALGVTACAGCTATTGGGGTCAAGGTACCCAGGTGACGGTCAG LVLAPGNVYSYWCTCTGCGCATCATGGAGGTAGCCCGGCAGGCTCTCCGA GQGTQVTVSSAHCCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCG HGGSPAGSPTSTEGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGG EGTSESATPESGPCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAAT GTSTEPSEGSAPGCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAG TSESATPESGPGSACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGC EPATSGSETPGTSACCAGGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGG TEPSEGSAPGSPAAAGGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCA GSPTSTEEGTSTEGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGG PSEGSAPGTSTEPTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGTA SEGSAPGTSTEPSCTTCTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTT EGSAPGTSTEPSECTACTGAACCTTCCGAAGGTAGCGCACCAGGTTCGTCT GSAPGTSTEPSEGTCACTCGAGGGTACCGAACTTTTCACTGGAGTTGTCCCA SAPGSSSLEGTELATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAA FTGVVPILVELDGATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACAT DVNGHKFSVSGEACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGA GEGDATYGKLTLAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTC KFICTTGKLPVPWTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCAC PTLVTTFSYGVQATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGA CFSRYPDHMKRHAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATG DFFKSAMPEGYVACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAA QERTISFKDDGNGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATT YKTRAEVKFEGDGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACT TLVNRIELKGIDFCGAGTACAACTATAACTCACACAATGTATACATCACGG KEDGNILGHKLECAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAA YNYNSHNVYITAAATTCGCCACAACATTGAAGATGGATCCGTTCAACTAG DKQKNGIKANFKCAGACCATTATCAACAAAATACTCCAATTGGCGATGGC IRHNIEDGSVQLACCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAA DHYQQNTPIGDGTCTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCA PVLLPDNHYLSTCATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTGG QSALSKDPNEKRTGGCTCTCATCACCATCACCATCACCATCACTAA DHMVLLEFVTAA GIGGSHHHHHHH H

Example 26: Purification of aIL6R-XTEN

E. coli from a single colony containing either: AC341, AC342, AC361, orAC362 were grown to saturation in 2×YT media. 10 mL of this saturatedovernight culture was used to inoculate a 500 ml of 2×YT media and thisculture was grown to an OD600 between 0.4 and 0.5 at 37° C., transferredto 26° C. and induced with 1 mM IPTG. The culture was then grownovernight (15-17 hours) and harvested by centrifugation at 10,000 rpm ina Sorvall SLA-3000 rotor. The pellets were stored until use at −80° C.Cell paste was resuspended in 25 ml of lysis buffer (20 mM sodiumacetate, 50 mM sodium chloride, pH 4.5), lysed by sonication, andclarified by spinning at 10,000 rpm at 4° C. in a Sorvall SS34 rotor.The samples were further acidified by the addition of acetic acid. Thesample was further clarified by centrifugation and the supernatantloaded onto a 15 mL DE52 column equilibrated with 20 mM sodium acetate,50 mM sodium chloride, pH 4.5. The column was washed with 2 columnsvolumes of 20 mM sodium acetate, 50 mM sodium chloride, pH 4.5, washedwith four columns volumes of 20 mM sodium acetate, 100 mM sodiumchloride, pH 4.5, and eluted with four columns volumes of 20 mM sodiumacetate, 150 mM sodium chloride, pH 4.5. Sodium sulfate was added to theelution fractions to a final concentration of 1M and the sample loadedonto a phenyl HIC column. The column was washed with five column volumesof 20 mM sodium acetate, 1M sodium sulfate pH 4.5, and eluted with fourcolumn volumes of 20 mM sodium acetate, 0.5M sodium sulfate pH 4.5. Thesamples were exchanged in to assay buffer, assigned lots numbers AP342,AP343, AP344 and AP345 and stored frozen at −80° C.

Example 27: Purification of aHER2-XTEN-GFP

AC62 was grown to saturation overnight in 2×YT+kanamycin. Two 500 mLflasks of 2×YT were inoculated with 3 ml of this saturated overnight andgrown to an OD600 of ˜0.8 at 37° C. The culture was brought to 25° C.and then induced with 1 mM IPTG. The culture was induced overnight. Thecell pellet was harvested by centrifugation at 5000 rpm, in a SorvallSLA-3000 at 4° C. as pellet EP52. The cell pellet was then resuspendedin 30 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole. The cells were lysedby sonication and then clarified by centrifugation at 15,000 rpm in aSorvall SS34 rotor. The supernatant was loaded onto a 5 ml Ni-NTAcolumn, washed with 20 column volumes of 30 mM Tris pH 8.0, 500 mM NaCl,20 mM imidazole, and eluted with 30 mM Tris pH 8.0, 500 mM NaCl, 600 mMimidazole. The elution fractions were diluted 1:2 with 20 mM histidinepH 5.6, and loaded onto an anion exchange column equilibrated with 20 mMhistidine pH 5.6. The column was washed with 20 mM histidine pH 5.6, 20column volumes of 20 mM histidine pH 5.6, 150 mM NaCl, 20 column volumesof 20 mM histidine pH 5.6, 300 mM NaCl, and then eluted with 20 mMhistidine pH 5.6, 600 mM NaCl. This protein was assigned lot #AP60 andstored frozen.

Example 28: Purification of aCD3-XTEN-GFP

AC50 was grown to saturation overnight in 2×YT+kanamycin. A 500 mL flaskof 2×YT was inoculated with 3 ml of this saturated overnight and grownto an OD600 of ˜0.8 at 37° C. The culture was brought to 25° C. and theninduced with 0.2 mM IPTG. The culture was induced overnight. The cellpellet was harvested by centrifugation at 5000 rpm, in a SorvallSLA-3000 at 4° C. The cell pellet was then resuspended in 30 mM Tris pH8.0, 500 mM NaCl, 20 mM imidazole. The cells were lysed by sonicationand then clarified by centrifugation at 15,000 rpm in a Sorvall SS34rotor. The supernatant was loaded onto Ni-NTA column, washed with 10column volumes of 30 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole, andeluted with 30 mM Tris pH 8.0, 500 mM NaCl, 600 mM imidazole. Thisprotein was assigned lot #AP43.

Example 29: Purification of aHER2-XTEN-aEGFR

AC49 was grown to saturation overnight in 2×YT+kanamycin. A 500 mL flaskof 2×YT was inoculated with 3 ml of this saturated overnight and grownto an OD600 of ˜0.8 at 37° C. The culture was brought to 25° C. and theninduced with 0.2 mM IPTG. The culture was induced overnight. The cellpellet was harvested by centrifugation at 5000 rpm, in a SorvallSLA-3000 at 4° C. as pellet EP36. The cell pellet was then resuspendedin 30 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole. The cells were lysedby sonication and then clarified by centrifugation at 15,000 rpm in aSorvall SS34 rotor. The supernatant was loaded onto a 5 ml Ni-NTAcolumn, washed with 10 column volumes of 30 mM Tris pH 8.0, 500 mM NaCl,20 mM imidazole, and eluted with 30 mM Tris pH 8.0, 500 mM NaCl, 600 mMimidazole. The elution fractions were diluted 1:3 with 20 mM histidinepH 5.6, and loaded onto a 1 mL DEAE column equilibrated with 20 mMhistidine pH 5.6, 200 mM NaCl. The column was washed with five columnvolumes of 20 mM histidine pH 5.6, 400 mM NaCl and five column volumesof 20 mM histidine pH 5.6, 600 mM NaCl, and then eluted with 20 mMhistidine pH 5.6, 700 mM NaCl. This protein was assigned lot #AP41. Thefinal purified aHER2-XTEN-aEGFR protein were subjected to non-reducingSDS-PAGE using NuPAGE 4-12% Bis-Tris gel from Invitrogen according tomanufacturer's specifications. The results, as shown in Lane 3 of FIG.17B (compared to aHER2-XTEN-aHER2-XTEN in Lane 2) demonstrate that theprotein was recovered at >95% purity by the process detailed above.Results of SEC analysis of the purified material, performed as describedin Example 36, are shown in FIG. 17C, and demonstrate that the bindingfusion protein was recovered in monomeric form and has an apparentmolecular weight of approximately 500 kDa, far in excess of its actualmolecular weight.

Example 30: Purification of aCD3-XTEN-aHER2

AC59 was grown to saturation overnight in 2×YT+kanamycin. A 10 L cultureof 2×YT in wavebag was inoculated with this saturated overnight andgrown to an OD600 of 1.3 at 37° C. The culture was brought to 25° C. andthen induced with 1 mM IPTG. The culture was induced overnight. The cellpellet was harvested by centrifugation at 5000 rpm, in a SorvallSLA-3000 at 4° C. and stored as pellet EP50. The cell pellet was thenresuspended in 20 mM Tris pH 8.0, 50 mM NaCl, plus Roche completeprotease inhibitors. The cells were lysed by sonication and thenclarified by centrifugation at 15,000 rpm in a Sorvall SSR-15 rotor. Thesupernatant was loaded onto a 60 ml Ni-NTA column, washed with 11 columnvolumes of 20 mM Tris pH 8.0, 50 mM NaCl; 10 column volumes of 20 mMTris pH 8.0, 500 mM NaCl; 10 column volumes of 20 mM Tris pH 8.0, 1%triton X-114; 10 column volumes of 20 mM Tris pH 8.0, 5 mM imidazole; 10column volumes of 20 mM Tris pH 8.0, 50 mM NaCl, 10 mM imidazole, andeluted with 20 mM Tris pH 8.0, 50 mM NaCl, 200 mM imidazole. The elutionfractions were diluted with 20 mM histidine pH 6.2, and triton-X114extracted to remove endotoxin as follows: bring to 5% detergent on ice,warm to 37° C. to cloud, phase separate by centrifugation at room tempat 3000 rpm in a Sorvall bench top centrifuge, transfer top aqueousphase, repeat process 3 times. The final aqueous layer was diluted with20 mM histidine pH 6.2 and loaded onto a 7.5 mL Q-sepharose FF columnequilibrated with 20 mM histidine pH 6.2, 20 mM NaCl. The column waswashed with 11 column volumes of 20 mM histidine pH 6.2, 20 mM NaCl, 10column volumes of 20 mM histidine pH 6.2, 50 mM NaCl, and 10 columnvolumes of 20 mM histidine pH 6.2, 100 mM NaCl, then eluted with 20 mMhistidine pH 5.6, 600 mM NaCl. The protein was then further purified byrunning a mono-Q column on an AKTA purifier system. The column andsystem were sanitized with 10 column volumes of 0.5 NaOH and the proteinloaded in 20 mM histidine pH 6.0 and eluted with a linear salt gradient.The viable fractions were pooled and triton extracted as above, tofurther reduce endotoxin. This protein was assigned lot #AP58.

Example 31: Purification of aHER2-XTEN-aHER2

AC47 was grown to saturation overnight in 2×YT+kanamycin. A 500 mL flaskof 2×YT was inoculated with 3 ml of this saturated overnight and grownto an OD600 of ˜0.8 at 37° C. The culture was brought to 25° C. and theninduced with 1 mM IPTG. The culture was induced overnight. The cellpellet was harvested by centrifugation at 5000 rpm, in a SorvallSLA-3000 at 4° C. and stored as EP35. The cell pellet was thenresuspended in 30 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole. Thecells were lysed by sonication and then clarified by centrifugation at15,000 rpm in a Sorvall SS34 rotor. The supernatant was loaded onto a 5ml Ni-NTA column, washed with 10 column volumes of 30 mM Tris pH 8.0,500 mM NaCl, 20 mM imidazole, and eluted with 30 mM Tris pH 8.0, 500 mMNaCl, 600 mM imidazole. The elution fractions were diluted 1:3 with 20mM histidine pH 5.6, and loaded onto a 1 mL DEAE column equilibratedwith 20 mM histidine pH 5.6, 200 mM NaCl. The column was washed withfive column volumes of 20 mM histidine pH 5.6, 400 mM NaCl and fivecolumn volumes of 20 mM histidine pH 5.6, 600 mM NaCl, and then elutedwith 20 mM histidine pH 5.6, 700 mM NaCl. This protein was assigned lot#AP40.

Example 32: Purification of Multivalent aEGFR Vhh Binders

Protein from constructs LCW501.001, LCW502.009, LCW503.004, LCW504.004,and LCW505.002 were expressed as follows: Nine 96 well plates with 0.5ml in each well were grown for each construct. Plates were filled byinoculating 40 ml of saturated overnight in SB into 1 L of autoinduction media and then using the Q-fill to load the plates. Thesamples were sealed with a breathable membrane and then placed at 26 Covernight shaking at 300 rpm. The plates were then pooled into one largebucket and poured into a single centrifuge bottle for harvesting. Thecultures were spun at 10,000 rpm in the Sorvall RC-5B centrifuge for 20mins (GS-3 rotor) to pellet the cells. Approximately 15 g of cell masswas obtained. The cells were resuspended in lysis buffer (20 mMphosphate pH 7.4, 500 mM NaCl, 5 mM imidazole, 0.5 mM EDTA and 1complete Roche protease mini tablet per 10 ml). The samples were thenlysed by sonication (5×20 seconds on, 40 seconds off at power setting 8with the large probe). The samples were kept on ice, but still warmed to˜25 during the process. The lysates were then spun at 15,000 rpm in theSS-34 rotor using a Sorvall RC-5B centrifuge to clarify.

2 mL Ni-NTA columns were equilibrated in 10 CV's of lysis buffer andthen the lysates from LCW503, LCW504 and LCW505 were loaded. The columnswere washed with 10 CV's of lysis buffer, 3 CV's of PBS+5 mM imidazoleto reduce salt, and then were eluted with PBS+300 mM imidazole. Theelution was tracked by GFP such that all of the fluorescence wascaptured in on fraction. The elutions were held overnight. The followingday 3×3 ml (LCW503, LCW504, LCW505) and 2×5 ml (LCW501 and LCW502)Ni-NTA columns were equilibrated in 10 CV's of lysis buffer and then thelysates from LCW503, LCW504 and LCW505 were loaded. The columns werewashed with 50 ml of lysis buffer, 15 ml of PBS+5 mM imidazole to reducesalt, and then were eluted with PBS+300 mM imidazole. The elution wastracked by GFP such that all of the fluorescence was captured in onfraction. The elutions from day one for 503, 504 and 505 were pooledwith the second day elutions.

The samples were then purified with anion exchange chromatography. A 1ml mono Q column was used for all of the preps. Buffer A was always 20mM Tris pH 8.0 and buffer B was always 20 mM Tris pH 8.0 and 1M NaCl.The AKTA purifier system was used with a 10 ml super loop. All runsinvolved loading with 12 ml injection (regardless of sample volume),washing with 2 CV's of buffer A, a gradient to 50% B over 20 CV's, andfinally Then a 5 CV washout with 100% B. The column was thenre-equilibrated with buffer A between runs. The super loop wasdisassembled and clean and the tubing flushed to avoid carry overbetween runs. The fractions were pooled and stored overnight at 4 C. Thenext day the samples concentrated in an Amicon ultra 30,000 MWCOconcentrator at 4000 rpm in the Sorvall T21. The concentrated sample wasloaded on gel filtration columns. The columns were TSK3000 for LCW501and LCW502 and TSK4000 for LCW503, LCW504 and LCW505. Fractions werepooled, concentrated and frozen for storage at −80° C.

Example 33: Characterization of aIL6R-XTEN

E. coli from a single colony of AC342 was grown to saturation in 2×YTmedia. 10 mL of this saturated overnight culture was used to inoculate a500 ml of 2×YT media and this culture was grown to an OD600 between 0.4and 0.5 at 37° C., transferred to 26° C. and induced with 1 mM IPTG. Theculture was then grown overnight (15-17 hours) and harvested bycentrifugation at 10,000 rpm in a Sorvall SLA-3000 rotor. The pelletswere stored until use at −80° C. Cell paste was resuspended in 25 ml oflysis buffer (20 mM sodium acetate, 50 mM sodium chloride, pH 4.5),lysed by sonication, and clarified by spinning at 10,000 rpm at 4° C. ina Sorvall SS34 rotor. The samples were further acidified by the additionof acetic acid. The sample was further clarified by centrifugation andthe supernatant loaded onto a 15 mL DE52 column equilibrated with 20 mMsodium acetate, 50 mM sodium chloride, pH 4.5. The column was washedwith 2 columns volumes of 20 mM sodium acetate, 50 mM sodium chloride,pH 4.5, washed with four columns volumes of 20 mM sodium acetate, 100 mMsodium chloride, pH 4.5, and eluted with four columns volumes of 20 mMsodium acetate, 150 mM sodium chloride, pH 4.5. Uniformity was assessedby SEC, which showed a monodispersed peak with minimal contamination(FIG. 28A). IL6R binding was assessed in an ELISA as follows: the plateswere coated with either 100 ng/well of human IL6R for 1 hour at 37° C.,blocked with 3% BSA in PBS for one hour at 37° C., bound by adding theAC342 dilution series to the plate and incubating for one hour at roomtemperature, washed 3 times with PBST, detected with biotinylatedanti-XTEN antibody for one hour at room temperature, washed three timeswith PBST, amplified with streptavidin-HRP for one hour at roomtemperature, washed three times with PBST, developed with TMB substrateand read at 405 nm using a plate reader. The ELISA values were plottedversus concentration (log scale), which provided determination of anEC50 value of 5 nM (FIG. 28B).

Example 34: Characterization of aCD40-XTEN

Two aCD40-XTEN constructs, AC384 and AC385, were expressed by growing aculture to saturation in 2×YT and then using this culture to inoculate a500 ml flask of 2×YT. This second flask was grown to an OD600 of between0.6 and 1.0 at 37° C., transferred to 26° C., and induced with 1 mMIPTG. The induction was left overnight and cell paste harvested bycentrifugation the flowing morning. These pellets were stored as EP220and EP221. Small samples were taken from the pellet, lysed usingbugbuster, and clarified using a microcentrifuge. These cleared lysateswere the serially diluted by a factor of three in PBST, for using in abinding ELISA. The ELISA was performed as follows: The plates werecoated with either 100 ng/well of human CD40-Fc fusion for 1 hour at 37°C., blocked with 3% BSA in PBS for one hour at 37° C., bound by addingthe AC384 or AC385 dilution series to the plate and incubating for onehour at room temperature, washed 3 times with PBST, detected withbiotinylated anti-XTEN antibody for one hour at room temperature, washedthree times with PBST, amplified with streptavidin-HRP for one hour atroom temperature, washed three times with PBST, developed with TMBsubstrate and read at 405 nm using a plate reader. The ELISA values wereplotted versus dilution of lysate plotted on a log scale and an EC50value determined, with nearly identical results (FIG. 27). Combiningthis with an estimate concentration of 10 μM in the lysate, theaCD40-XTEN constructs have a Kd of approximately 50 nM for human CD40.

Example 35: Characterization of aHER2-XTEN-aHER2

Binding of the aHER2-XTEN-aHER2 protein to human HER2 was assessed byELISA as follows: The plates were coated with either 100 ng/well ofhuman HER2-Fc fusion overnight at 4° C., blocked with 1% BSA in PBS forone hour at room temperature, bound by adding a dilution series ofaHER2-XTEN-aHER2 to the plate and incubating for one hour at roomtemperature, washed 3 times with PBST, detected with anti-HA antibodywith HRP conjugation for one hour at room temperature, washed threetimes with PBST, washed three times with PBST, developed with ABTSsubstrate and read at 405 nm using a plate reader. The ELISA values wereplotted versus concentration plotted on a log scale and an EC50 of 0.5nM determined (FIG. 18).

Example 36: Characterization of aCD3-XTEN-aHER2

To confirm the binding specificity of expressed and purified bindingfusion proteins with scFv targeting moieties, two preparations directedto the targets HER2 or CD3 were evaluated. Binding fusion proteins ofaHER2 linked to the N-terminus of XTEN, with GFP linked to theC-terminus of the XTEN (“aHER2-XTEN-GFP”), and aCD3 linked to theN-terminus of XTEN, with GFP linked to the C-terminus of the XTEN(“aCD3-XTEN-GFP”) were the test articles that were evaluated for theirability to bind their respective targets on Jurkat cells bearing CD3 andSK-BR-3 cells bearing HER2. Jurkat cells and SK-BR3 cells were incubatedwith the indicated scFv-XTEN-GFP fusion proteins. Bound aCD3-XTEN-GFPand aHER2-XTEN-GFP were detected with a polyclonal anti-GFP antibodyspecific for the GFP fused to the construct, and an appropriateFITC-conjugated secondary antibody using flow cytometry. The flowcytometry results, shown graphically in FIG. 19B, demonstrate specificbinding of the aCD3-XTEN-GFP to Jurkat cells and specific binding of theaHER2-XTEN-GFP to SK-BR-3 cells, respectively, while there was nonon-specific binding detected in the opposite configuration, indicatinglack of cross-reactivity by the constructs.

A bispecific binding fusion protein with scFv targeting moieties to bothHER2 and CD3 (“aCD3-XTEN-aHER2”, matching the N- to C-terminusconfiguration of the fusion protein components) was recovered by thepurification process detailed above and the protein characterized. TheaCD3-XTEN-aHER2 protein was subjected to non-reducing SDS-PAGE usingNuPAGE 4-12% Bis-Tris gel from Invitrogen according to manufacturer'sspecifications. The results, shown in FIG. 20A demonstrate that theprotein was >95% pure, as judged by SDS-PAGE. FIG. 20B shows the outputof a size exclusion chromatography (SEC) analysis of the aHER2-aCD3-XTENcompared to molecular weight standards, and demonstrates that no dimersor other higher-order oligomers are formed and that the protein has anapproximate apparent molecular weight of approximately 500 kDa,approximately five-fold higher than that derived from the SDS-PAGEassay.

An ELISA assay to detect direct binding of the aCD3-XTEN-aHER2 toHER2-Fc-coated wells was performed as follows. The extracellular domainof HER2 fused to the Fc domain of human IgG was coated on to the wellsof a microtiter plate. A dilution series of the aCD3-XTEN-aHER2 was thenapplied to the coated wells. After two hours the unbound aCD3-XTEN-aHER2was washed away and any bound protein detected with an HRP-conjugatedanti-HA antibody. The results of the binding assays are shown in FIG.21A. The control fusion protein with N-terminal aHER2 binds to HER2-Fcwith an apparent Kd of 80 pM, while the C-terminal fusion of theaCD3-XTEN-aHER2 binds with an apparent Kd of 3.4 nM. Thus, the anti-Her2component of the aCD3-XTEN-aHER2 construct still retains good bindingaffinity, although it is lower than the N-terminal construct.

Flow cytometry analysis of aHER2-XTEN (FIG. 19A-FIG. 19B) andaCD3-XTEN-aHER2 (FIG. 21C) show binding to HER2-expressing SK-BR-3cells. Detection was via an anti-HA antibody and appropriate secondaryantibody. The binding of aCD3-XTEN-GFP to CD3-positive Jurkat cells wasmeasured by flow cytometry, using anti-GFP antibody detection, in thepresence of a 10-fold molar excess of aHER2-aCD3-XTEN. The resultsclearly demonstrate that excess bispecific aCD3-XTEN-aHER2 competitivelydisplaces the monospecific aCD3-XTEN-GFP protein and eliminates theobserved MFI shift (FIG. 22). Thus, we have engineered a aCD3-XTEN-aHER2bispecific binding fusion protein that interacts with HER2-expressingtumor cells and CD3-positive T-cells.

To demonstrate the ability of the aCD3-XTEN-aHER2 binding fusion proteinto target and kill cancer cells, an assay was performed using theconstruct to direct the cytotoxic activity of T-cells towards tumorcells. Here, we tested the efficacy of the candidate aCD3-XTEN-aHER2using M21 melanoma and SK-BR-3 breast cancer cells. Tumor cells,expressing the target receptor, were labeled with a membrane permeablefluorophore, DiOC₁₈. Fresh peripheral blood mononuclear cells (PBMCs)were then mixed with the labeled tumor cells (Effector cell:Tumor cellratio 10:1) and the aCD3-XTEN-aHER2 binding fusion protein. After 24hours the cell suspensions were collected by centrifugation and deadcells were labeled with propidium iodide. The cells were analyzed on aZeiss Axiovert 100 microscope. Images of target (DiOC₁₈) and dead cells(propidium iodide) were captured with a SPOT CCD camera usingappropriate excitation and emission filter sets. Image analysis wasconducted using the program ImageJ. The number of target cells per imagewas determined by using the analyze particles command using the green(DiOC₁₈) fluorescence channel image. The fraction of these cells thatwere dead was determined by overlapping the red (propidium iodide)fluorescence channel using the co-localization plugin and counting thenumber green cells that were also red. The proportion of dead tumorcells, as a function of aHER2-aCD3-XTEN concentration is shown in FIG.23. A general linear model with a logit link function was fit to thedata with concentration of aCD3-XTEN-aHER2 fusion protein as theindependent variable. The model found aCD3-XTEN-aHER2 concentration tobe a significant predictor of tumor cell death at a p-value of <0.001for both M21 and SK-BR-3 target cells. The results support theconclusion that binding fusion proteins with targeting moieties directedto tumor associated antigens can have activity against tumor cells.

Example 37: Characterization of aHER2-XTEN-aEGFR

Size exclusion chromatography was performed on recoveredaHER2-XTEN-aEGFR to assess its monomeric characteristics. Results fromthe SEC analysis demonstrate that the aHER2-XTEN-aEGFR (FIG. 17C) didnot dimerize or form other higher order oligomers. As shown in FIG. 17C,the apparent molecular weight is approximately five-fold larger than itscalculated mass (approximately 107 kDa) or the mass estimated from theSDS PAGE of FIG. 17B, due to the XTEN component; here the elution volumeof aHER2-XTEN-aEGFR is compared a set of globular molecular weightstandards (from left to right: 670, 158, 44, 17, and 1.4 kDa). Thebinding activity of aHER2-XTEN-aEGFR to its respective targets wastested in a ELISA format. The extracellular domains of either HER2 orEGFR fused to the Fc domain of human IgG were coated on to the wells ofa microtiter plate. A dilution series of the aHER2-XTEN-aEGFR was thenapplied to the coated wells. After two hours the unboundaHER2-XTEN-aEGFR was washed away and any bound protein detected with anHRP-conjugated Anti-HA antibody. The result indicate that thebifunctional BPXTEN construct was able to bind each target atessentially equivalent concentrations. (FIG. 18).

Example 38: Characterization of Multivalent aEGFR Vhh Binding FusionProteins

Characterization of domain binding fusion proteins was performed onaEGFR-XTEN Vhh constructs that had increasing numbers of targetingmoieties, as depicted schematically in FIG. 24A. SDS-PAGE and SEC wereperformed with four constructs; LCW501.001, LCW502.009, LCW503.004, andLCW504.004. A clear laddering across constructs in the SDS-PAGEanalysis, shown in FIG. 24B, demonstrates the increasing length of theprotein as the number of anti-EGFR binding domains increases. This ismirrored by the SEC that, in FIG. 25, shows an increase in thehydrodynamic radius of the proteins as the number of anti-EGFR domainsincreases. The derived values for apparent molecular weight factor,apparent molecular weight factor and hydrodynamic radii are presented inTable 26. Note that these hydrodynamic radii are characteristicallylarge for a protein of this molecular weight due to the unstructurednature of the XTEN linker domains, resulting in an increased apparentmolecular weight for each of the domain binding fusion proteins.

LCW501.001, LCW502.009, LCW503.004, and LCW504.004 were alsocharacterized in two different ELISA experiments. In the first Costar3690 plates were coated with 100 ng of EGFR-Fc in 50 μl of PBS overnightat 4° C., blocked with 3% BSA in PBS for 1 hour, bound with multivalentaEGFR Vhh binders from 1 μM downward using 5-fold dilutions in bindingbuffer (1% BSA in PBST) for 2 hours at RT with shaking at 80 rpm, washedthree times with PBST, detected with a 1:5000 dilution of goatanti-GFP-HRP for 1 hour at room temperature with shaking at 80 rpm,washed five times with PBST, developed with ABTS-H₂O₂ substrate and readat 405 nm. Binding curves were determined for each of the differentmultimer species. All of the higher order multimers showed tighterbinding than the monomer indicating an avidity effect and that multiplebinding sites were active in the multivalent molecules (FIG. 26A). Thesecond ELISA based characterization was as follows: Costar 3690 platewere coated with 200 ng of goat anti-GFP in 50 μl of PBS overnight at 4°C., blocked with 3% BSA in PBS for 1 hour, bound with multivalent aEGFRVhh binders from 3 μM downward using 5-fold dilutions in binding buffer(1% BSA in PBST) for 2 hours at RT with shaking at 80 rpm, washed threetimes with PBST, detected with 100 ng/well (˜20 nM) of biotinylatedEGFR-Fc for 1 hour at room temperature with shaking at 80 rpm, washedthree times with PBST, amplified with 1:5000 dilution ofstreptavidin-HRP in binding buffer and developed with ABTS-H2O2substrate and read at 405 nm. The higher order multimers showed andincreased capacity for EGFR-Fc binding (FIG. 26B) indicating thatincreasing the number of EGFR binding modules in the protein increasesthe number of available binding sites within the protein. These datasuggest that all of the binding sites in the binding fusion proteins,including the higher order tetramers and hexamers, are active andcapable of binding EGFR.

Example 39: Purification of aHER2-XTEN

AC51 was grown to saturation overnight in 2×YT+kanamycin. A 500 mL flaskof 2×YT was inoculated with 3 ml of this saturated overnight and grownto an OD600 of ˜0.8 at 37° C. The culture was brought to 25° C. and theninduced with 0.2 mM IPTG. The culture was induced overnight. The cellpellet was harvested by centrifugation at 5000 rpm, in a SorvallSLA-3000 at 4° C. The cell pellet was then resuspended in 30 mM Tris pH8.0, 500 mM NaCl, 20 mM imidazole. The cells were lysed by sonicationand then clarified by centrifugation at 15,000 rpm in a Sorvallcentrifuge. The supernatant was loaded onto Ni-NTA column, washed with10 column volumes of 30 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole,and eluted with 30 mM Tris pH 8.0, 500 mM NaCl, 600 mM imidazole. Thisprotein was assigned lot #AP44.

Example 40: Analytical Size Exclusion Chromatography of XTEN FusionProteins

Size exclusion chromatography analysis was performed on binding fusionproteins containing various targeting moieties and unstructuredrecombinant proteins of varying length to determine the effect on XTENon increasing the apparent molecular weight. An exemplary assay used aTSKGel-G4000 SWXL (7.8 mm×30 cm) column in which 40 μg of purifiedglucagon fusion protein at a concentration of 1 mg/ml was separated at aflow rate of 0.6 ml/min in 20 mM phosphate pH 6.8, 114 mM NaCl.Chromatogram profiles were monitored using OD214 nm and OD280 nm. Columncalibration for all assays were performed using a size exclusioncalibration standard from BioRad; the markers include thyroglobulin (670kDa), bovine gamma-globulin (158 kDa), chicken ovalbumin (44 kDa),equine myoglobuin (17 kDa) and vitamin B12 (1.35 kDa). RepresentativeSEC profiles of binding fusion proteins are shown as overlays in FIGS.17C, 20 and 25. Based on the SEC analyses for all constructs evaluated,the apparent molecular weight factors, the apparent molecular weightfactor (expressed as the ratio of apparent molecular weight factor tothe calculated molecular weight) and the hydrodynamic radius (R_(H) innm) are shown in Table 26. The data show that the apparent molecularweight of each compound is proportional to the length of the attachedXTEN sequence. This is particularly evident in the case of the EGFR Vhhconstructs that included increasing units of EGRF_VHH-AM144_XTEN, goingfrom monomer to dimer to tetramer to hexamer, and showed increases inapparent molecular weight factor as the cumulative length of XTENincreased with each addition of the AM144 linkers. The data also showthat the apparent molecular weight of each construct is significantlylarger than that expected for a globular protein (as shown by comparisonto the standard proteins run in the same assay). Additionally, theincorporation of XTEN fusion partners with 244 total amino acids or moreinto fusion proteins with targeting moieties resulted with ahydrodynamic radius of 7 nm or greater; well beyond the glomerular poresize of approximately 3-5 nm. Accordingly, it is concluded that bindingfusion proteins comprising targeting moieties and XTEN would havereduced renal clearance, contributing to increased terminal half-lifeand improving the therapeutic or biologic effect relative to acorresponding un-fused targeting moiety proteins.

TABLE 26 SEC analysis of various polypeptides Apparent XTEN or ActualApparent Molecular fusion Therapeutic MW MW Weight R_(H) Construct Namepartner Protein (kDa) (kDa) Factor (nm) XTEN_AE912- AE912 Anti-IL6R 111883 7.9 8.7 aIL6R scFv aHER2- Y288 Anti-HER2 83 419 5.0 7.4 XTEN_Y288-aHER2 EGFR_Vhh- AM144 Anti-EGFR 54 107 2 4.9 XTEN monomer EGFR_Vhh-AM144 X2 Anti-EGFR 81 329 4.1 7.0 XTEN dimer EGFR_Vhh- AM144 X4Anti-EGFR 135 1022 7.5 9.1 XTEN tetramer EGFR_Vhh- AM144 X6 Anti-EGFR189 1802 9.5 10.1 XTEN hexamer

Example 41: Creation, Purification and Characterization of BindingFusion Protein-Conjugates-aHer2-XTEN Constructs with Conjugated AF680Fluorophore

To test the feasibility and utility of binding fusion protein-drugconjugates, binding fusion proteins were conjugated with a fluorophorethat was then characterized for binding affinity and utilized inpreclinical animal models to evaluate the ability of the conjugates tosystemically distribute after injection and bind target tumor tissue.

aHer2-XTEN(AE864-Cys)-AF680

The E. coli containing the AC452 gene on a plasmid were grown in liquidculture to saturation overnight and then 90 ml of this culture was usedto inoculate 4.5 L pho induction media, divided evenly between 9 4 Lflasks. Cultures were grown at 26° C. in the presence of 10 μg/mltetracycline and were induced as phosphate was depleted from the media.Protein was trafficked to the periplasm of the host cells via an STIIsignal sequence fused to the N-terminus of the encoded protein that wassubsequently removed by post-translational modification in the cells.The cell pellet was harvested by centrifugation for 20 min at 4000 rpmin a SLA-3000 rotor. Some of the cell pellet (45 of 90 total g) wasresuspended in 121 ml of PBS plus 10 mM imidizole and 13.5 ml ofBugBuster and DNase were added. The cells were lysed by vortexingperiodically over a 90 minute period. The lysate was then clarified bycentrifugation at 15,000 rpm for 30 minutes. The clarified lysate wasthen loaded on to an 85 ml toyopearl chelate column charged with 100 mMNiSO4, and equilibrated with PBS plus 10 mM imidizole. The column waswashed with 5 column volumes of PBS plus 10 mM imidizole, and theprotein eluted with 3 column volumes of PBS plus 250 mM imidizole andthen stripped with 1.2 column volumes of PBS plus 500 mM imidizole. Theeluate was reduced with 0.3 mM TCEP and then labeled for 3 hours at roomtemperature with Alexa Fluor 680 at a ratio of 0.1 mg of dye per mg ofprotein. The sample was then transferred to 4° C. for overnight storage.The sample was then diluted 2.7 fold with water to reduce theconductivity and loaded on to a macrocapQ column, previously sanitizedwith NaOH and equilibrated with 20 mM Tris pH 7.5, 50 mM NaCl. Thecolumn was washed with 5 column volumes of 20 mM Tris pH 7.5, 50 mM NaCland eluted with a 10 column volume linear gradient from 150 mM NaCl to300 mM NaCl both with 20 mM Tris pH 7.5 as the buffer. The pooledelution fractions were concentrated using an Amicon ultra concentratorwith a 10,000 MWCO membrane and stored at −80° C., assigned lot #AP502.

aHer2-XTEN(AE576-Cys)—AF680

The E. coli containing the AC451 gene on a plasmid were grown in liquidculture to saturation overnight and then 90 ml of this culture was usedto inoculate 10 L pho induction media, divided evenly between 20 4 Lflasks. Cultures were grown at 26° C. in the presence of 10 μg/mltetracycline and were induced as phosphate was depleted from the media.Protein was trafficked to the periplasm of the host cells via an STIIsignal sequence fused to the N-terminus of the encoded protein that wassubsequently removed by post-translational modification in the cells.The cell pellet was harvested by centrifugation for 20 min at 4000 rpmin a SLA-3000 rotor. The cell pellet (39 g) was resuspended in 96.3 mlof PBS plus 10 mM imidizole and 10.7 ml of BugBuster and DNase wereadded. The cells were lysed by vortexing periodically over a 55 minuteperiod. The lysate was then clarified by centrifugation at 15,000 rpmfor 25 minutes. The clarified lysate was then loaded on to an 85 mltoyopearl chelate column charged with 100 mM NiSO4, and equilibratedwith PBS plus 10 mM imidizole. The column was washed with 5 columnvolumes of PBS plus 10 mM imidizole, and the protein eluted with 3column volumes of PBS plus 250 mM imidizole and then stripped with 1.2column volumes of PBS plus 500 mM imidizole. The eluate was reduced with0.3 mM TCEP and then labeled for 3 hours at room temperature with AlexaFluor 680 at a ratio of 0.1 mg of dye per mg of protein. The sample wasthen transferred to 4° C. for overnight storage. The sample was thendiluted 2.7 fold with water to reduce the conductivity and loaded on toa macrocapQ column, previously sanitized with NaOH and equilibrated with20 mM Tris pH 7.5, 50 mM NaCl. The column was washed with 5 columnvolumes of 20 mM Tris pH 7.5, 50 mM NaCl and eluted with a 10 columnvolume linear gradient from 150 mM NaCl to 300 mM NaCl both with 20 mMTris pH 7.5 as the buffer. The pooled elution fractions wereconcentrated using an Amicon ultra concentrator with a 10,000 MWCOmembrane and stored at −80° C., assigned lot #AP486.

aHer2-XTEN(AE288-Cys)-AF680

The E. coli containing the AC450 gene on a plasmid were grown in liquidculture to saturation overnight and then 90 ml of this culture was usedto inoculate 10 L pho induction media, divided evenly between 20 4 Lflasks. Cultures were grown at 26° C. in the presence of 10 μg/mltetracycline and were induced as phosphate was depleted from the media.Protein was trafficked to the periplasm of the host cells via an STIIsignal sequence fused to the N-terminus of the encoded protein that wassubsequently removed by post-translational modification in the cells.The cell pellet was harvested by centrifugation for 60 min at 4000 rpmin a SLA-3000 rotor. The cell pellet (36 g) was resuspended in 97 ml ofPBS plus 10 mM imidizole and 10.7 ml of BugBuster and DNase were added.The cells were lysed by vortexing periodically over a 55 minute period.The lysate was then clarified by centrifugation at 15,000 rpm for 25minutes. The clarified lysate was then loaded on to an 85 ml toyopearlchelate column charged with 100 mM NiSO4, and equilibrated with PBS plus10 mM imidizole. The column was washed with 5 column volumes of PBS plus10 mM imidizole, and the protein eluted with 3 column volumes of PBSplus 250 mM imidizole and then stripped with 1.2 column volumes of PBSplus 500 mM imidizole. The eluate was reduced with 0.3 mM TCEP and thenlabeled for 3 hours at room temperature with Alexa Fluor 680 at a ratioof 0.1 mg of dye per mg of protein. The sample was then transferred to4° C. for overnight storage. The sample was then diluted 2.7 fold withwater to reduce the conductivity and loaded on to a macrocapQ column,previously sanitized with NaOH and equilibrated with 20 mM Tris pH 7.5,50 mM NaCl. The column was washed with 5 column volumes of 20 mM Tris pH7.5, 50 mM NaCl and eluted with a 10 column volume linear gradient from150 mM NaCl to 300 mM NaCl both with 20 mM Tris pH 7.5 as the buffer.The pooled elution fractions were concentrated using an Amicon ultraconcentrator with a 10,000 MWCO membrane and stored at −80° C., assignedlot #AP481.

SEC Analysis of aHer2-XTEN(AE288-Cys)-AF680,aHer2-XTEN(AE576-Cys)-AF680, aHer2-XTEN(AE576-Cys)-AF680

Confirmation that the Alexa Fluor 680 was conjugated to the XTENproteins was provided by analytical size exclusion chromatography. Thecharacteristic 690 nm absorbance of the dye would elute very late in theSEC chromatogram if it were free in solution, whereas if it wereconjugated to the XTEN protein it would elute earlier at thecharacteristic volume of the XTEN. The three constructs were assayedusing a BioSep 54000 (7.8×6000 mm, phenomenex) run on an Akta purifiersystem and the chromatogram for each of the three constructs wasmonitored at 280 (FIG. 33, solid lines) and 690 nm (FIG. 33, dottedlines). For each of the all three constructs, the 690 nm absorbancepeaks co-eluted with the 280 nm peak, indicating that the Alex Fluor 680was conjugated to the XTEN fusion protein. No free dye was noted at theexpected elution time of approximately 52 minutes. The chromatogramsalso reflect elutions proportional to the increase in apparent masscontributed by the length of the respective XTEN components.

Flow Cytometry Analysis of aHer2-XTEN Conjugates

Specific binding of a binding fusion protein to a tumor cell line wasevaluated using the constructs of anti-HER2 targeting moiety linked todifferent lengths of XTEN, with a fluorophore AF680 covalently linked toXTEN, as described above. The aHer2-XTEN-Cys-AF680 constructs binding toHer2 were assessed by flow cytometry on the Her2+ tumor cell line,SKOV3, as follows: SKOV3 cells were grown from frozen stocks andpassaged 2-3 times. Cells were resuspended into 31 tubes and incubatedat 1×10⁶ cells/tube with Fc block (BD BioSciences) for 10 minutes onice. Fifteen tubes were preincubated with 1000 nM unlabeled Herceptin(to block binding as a control) for 20 minutes on ice. All tubes (15incubated with unlabeled Herceptin, 15 not incubated with unlabeledHerceptin) were then incubated with the 100 nM of the appropriatelylabeled aHer2-XTEN-AE-288-Cys-AF680, aHer2-XTEN-AE-576-Cys-AF680, andaHer2-XTEN-AE-864-Cys-AF680 conjugate or the labeled isotype control for20 minutes on ice, washed twice with flow buffer, the pellets wereresuspended in 0.2 mL of flow buffer and then were run on an Accuro C6Flow Cytometer. Data were collected for forward and side scatter andside scatter vs. FL4 for Alexa680. Data (FIG. 34A-FIG. 34C) arepresented as histograms of each aHer2-XTEN-AE-864-Cys-AF680 (FIG. 34A),aHer2-XTEN-AE-576-Cys-AF680 (FIG. 34B), and aHer2-XTEN-AE-288-Cys-AF680conjugate (FIG. 34C) overlayed with blocked and unblocked Herceptin. Thedata show that Herceptin completely blocked the binding of theaHer2-XTEN-AE-288-Cys-AF680, aHer2-XTEN-AE-576-Cys-AF680, andaHer2-XTEN-AE-864-Cys-AF680 conjugate to Her2+ SKOV3 cells in vitro,demonstrating the specific binding of the conjugate constructs for theHer2 target on the tumor cells.

In Vivo and Ex Vivo Imaging of aHer2-XTEN Conjugates

Targeting and biodistribution of the aHer2-XTEN-Cys-AF680 constructs,prepared as described above, to Her2+ tumor was assessed using in vivo,followed by ex vivo, fluorescence imaging. Control groups included miceinjected with fluorescently tagged Herceptin-Alexa 680 and mice injectedwith aHer2-XTEN-864-Alexa 680 but blocked with Herceptin one hour priorto injection.

Female nu/nu mice bearing SKOV3 tumor cells were given a singleinjection of high or low dose aHer2-XTEN-AE-288-Cys-AF680,aHer2-XTEN-AE-576-Cys-AF680, aHer2-XTEN-AE-864-Cys-AF680 orHerceptin-AF680 control. Whole body scans were acquired pre-injectionand then at approximately 8, 24, 48 and 72 hours post-injection.Following the 72 hour time point all high dose groups were euthanizedand tumors, liver, lung, heart, spleen and kidneys were ex vivo imagedusing ex vivo fluorescence imaging. Fluorescence imaging was performedusing an IVIS 50 optical imaging system (Caliper Life Sciences,Hopkinton, Mass.). Cy5.5 excitation (615-665 nm) and emission (695-770nm) filters were selected to match the fluorescence agents' wavelengthsSmall and medium binning of the CCD chip was used and the exposure timewas between 5-20 seconds to obtain at least several thousand counts fromthe signals that were observable in each mouse in the image and to avoidsaturation of the CCD chip. To normalize images for quantification, abackground fluorescence image was acquired using background excitationand emission filters for the Cy5.5 spectral region.

In vivo imaging data are shown in FIG. 35 and Table 27. Several of thegroups showed specific fluorescent signals in the tumor, above the levelof the autofluorescent background. Significant signals were evident inmost of the higher dosage level (6.7 nmol/mouse) aHer2-XTEN test agentgroups (Groups 1, 3 and 5) as well as the positive control group dosedwith tagged herceptin (Group 7). In addition, there were minor trendssuggesting detection of the agents in the tumors for some of the groupswhere the lower dosage levels were administered. The data also showed a50% higher peak value for aHer2-XTEN-864-Alexa 680 (Group 1), comparedwith aHer2-XTEN-576-Alexa 680 (Group 3) and aHer2-XTEN-288-Alexa 680(Group 5). While the tagged herceptin showed peak tumor binding 8 hpost-administration, the test agents generally showed later peak bindingapproximately 24-48 h post-administration. aHer2-XTEN-288-Alexa 680showed the most rapid targeting kinetics, as well as the most rapidclearance.

Ex vivo imaging data shown in FIG. 36 and Table 28 summarizes the meantotal fluorescence signals measured by group and tissue type. Imaging oftumors in Groups 2 and 9 demonstrated approximately 6-fold higher totalfluorescence signal in Group 2 (0.67 nmol aHer2-XTEN-864-Alexa 680)compared with Group 9 (0.67 nmol aHer2-XTEN-864-Alexa 680+100× excessunlabeled Herceptin administered 1 hour prior) indicating specific Her2+tumor targeting. Results from the extended tissue set (tumor, heart,lungs, spleen, liver, kidneys) indicated that Group 7 (3.3 nmolHerceptin-Alexa680) showed the greatest signals in most tissues, butthat Group 1 (6.7 nmol aHer2-XTEN-864-Alexa 680), with the longest XTENmolecule, showed nearly comparable total signals in most cases exceptfor the liver, in which the Herceptin Group 7 had approximately doublethe fluorescent signal compared to the aHer2-XTEN-864 Group 1. Group 3(6.7 nmol aHer2-XTEN-576-Alexa 680) and Group 5 (6.7 nmolaHer2-XTEN-864-Alexa 680) showed lower signals in all tissues thanGroups 1 and 7.

TABLE 27 In vivo fluorescence by group of mean peak signals and signalat 72 h Average Fluorescence Efficiency (group Dosage mean normalized toLevel pre-treatment values) (nmol/ Peak Level Group # Test Materialmouse) Peak Time at 72 h Group 1 aHer2-XTEN-864- 6.7 20.3 24 h 11.2Alexa 680 Group 2 aHer2-XTEN-864- 0.67 5.3 48 h 3.5 Alexa 680 Group 3aHer2-XTEN-576- 6.7 14.0 24 h 6.9 Alexa 680 Group 4 aHer2-XTEN-576- 0.673.6 48 h 3.4 Alexa 680 Group 5 aHer2-XTEN-288- 6.7 13.4 8 h 4.2 Alexa680 Group 6 aHer2-XTEN-288- 0.67 3.5 48 h 3.1 Alexa 680 Group 7Herceptin-Alexa680 3.3 40.3 8 h 18.6 Control Group 8 Herceptin-Alexa6800.33 5.3 48 h 4.4 Control Group 9 aHer2-XTEN-864- 0.67 + 100x 6.0 72 h6.0 Alexa 680 + excess 1 h unlabeled Herceptin before

TABLE 28 Summary by group of mean total signals in tissues imaged exvivo. Total Fluorescence Efficiency Dosage Level (group mean) (×10⁶)Group # Test Material (nmol/mouse) Tumor Heart Lungs Spleen LiverKidneys Group 1 aHer2-XTEN- 6.7 42 28 130 16 180 120 864-Alexa 680 Group2 aHer2-XTEN- 0.67 4.2 — — — — — 864-Alexa 680 Group 3 aHer2-XTEN- 6.727 6.8 24 3.4 48 31 576-Alexa 680 Group 5 aHer2-XTEN- 6.7 7.2 1.9 5.62.1 20 34 288-Alexa 680 Group 7 Herceptin- 3.3 69 32 150 25 370 110Alexa680 Control Group 9 aHer2-XTEN- 0.67 + 100x 0.7 — — — — — 864-Alexa680 + excess 1 h unlabeled before Herceptin

Example 42: Pharmacokinetic Analysis of CTLA4-XTEN

The in vivo pharmacokinetics of CTLA4-XTEN constructs can be assessedusing standard methods for protein compositions. Pharmacokinetics isassessed in multiple species, however mice, rats, cynomolgus monkeys,and dogs are preferred due to their common usage in predicting humanpharmacokinetics. Compositions of CTLA4-XTEN constructs, or CTLA4 as acomparator, are typically provided in an aqueous buffer compatible within vivo administration (for example: phosphate-buffered saline orTris-buffered saline). The compositions would be administered atappropriate doses and via multiple routes: most preferably viaintravenous or subcutaneous routes. Blood samples would collected atappropriate time points ranging from 0.08 to 504 hours, and processedinto plasma. Plasma samples can be analyzed for concentration of testarticles by ELISA assay. Analysis is typically performed using asandwich ELISA format. Rabbit polyclonal anti-XTEN antibodies oranti-targeting moiety are coated onto wells of an ELISA plate. The wellsare blocked, washed and plasma samples are then incubated in the wellsat varying dilutions to allow capture of the compound by the coatedantibodies. Wells are then washed extensively, and bound proteindetected using a biotinylated preparation of a polyclonal anti CTLA4antibody or anti-XTEN antibody and streptavidin HRP. Concentrations oftest article are then calculated at each time point by comparing thecolorimetric response at each serum dilution to a standard curve.Pharmacokinetic parameters are then calculated using the WinNonLinsoftware package. It is expected that the results would support thefinding that addition of an XTEN to CTLA4 can greatly increase theterminal half-life compared to the targeting moiety not linked to XTEN,and enhance other pharmacokinetic properties as well.

Example 43: Pharmacokinetic Analysis of IL6R-XTEN

The in vivo pharmacokinetics of IL6R-XTEN constructs can be assessedusing standard methods for protein compositions. Pharmacokinetics isassessed in multiple species, however mice, rats, cynomolgus monkeys,and dogs are preferred due to their common usage in predicting humanpharmacokinetics. Compositions of IL6R-XTEN constructs, or IL6R ascomparator, are typically provided in an aqueous buffer compatible within vivo administration (for example: phosphate-buffered saline orTris-buffered saline). The compositions would be administered atappropriate doses and via multiple routes: most preferably viaintravenous or subcutaneous routes. Blood samples would be collected atappropriate time points ranging from 0.08 to 504 hours, and processedinto plasma. Plasma samples are analyzed for concentration of testarticles by ELISA assay. Analysis is typically performed using asandwich ELISA format. Rabbit polyclonal anti-XTEN antibodies orantibodies to the targeting moiety are coated onto wells of an ELISAplate. The wells are blocked, washed and plasma samples are thenincubated in the wells at varying dilutions to allow capture of thecompound by the coated antibodies. Wells are then washed extensively,and bound protein detected using a biotinylated preparation of apolyclonal anti-IL6R antibody or anti-XTEN antibody and streptavidinHRP. Concentrations of test article are then calculated at each timepoint by comparing the colorimetric response at each serum dilution to astandard curve. Pharmacokinetic parameters are then calculated using theWinNonLin software package. It is expected that the results wouldsupport the finding that addition of an XTEN to IL6R can greatlyincrease the terminal half-life compared to the targeting moiety notlinked to XTEN, and enhance other pharmacokinetic parameters, as well.

Example 44: Pharmacokinetic Analysis of CD40-XTEN

The in vivo pharmacokinetics of CD40-XTEN constructs can be assessedusing standard methods for protein compositions. Pharmacokinetics isassessed in multiple species, however mice, rats, cynomolgus monkeys,and dogs are preferred due to their common usage in predicting humanpharmacokinetics. Compositions of CD40-XTEN constructs, or CD40 ascomparator, are typically provided in an aqueous buffer compatible within vivo administration (for example: phosphate-buffered saline orTris-buffered saline). The compositions would be administered atappropriate doses and via multiple routes: most preferably viaintravenous or subcutaneous routes. Blood samples would be collected atappropriate time points ranging from 0.08 to 504 hours, and processedinto plasma. Plasma samples are analyzed for concentration of testarticles by ELISA assay. Analysis is typically performed using asandwich ELISA format. Rabbit polyclonal anti-XTEN antibodies orantibodies to targeting moiety are coated onto wells of an ELISA plate.The wells are blocked, washed and plasma samples are then incubated inthe wells at varying dilutions to allow capture of the compound by thecoated antibodies. Wells are then washed extensively, and bound proteindetected using a biotinylated preparation of a polyclonal anti CD40antibody or anti-XTEN antibody and streptavidin HRP. Concentrations oftest article are then calculated at each time point by comparing thecolorimetric response at each serum dilution to a standard curve.Pharmacokinetic parameters are then calculated using the WinNonLinsoftware package. It is expected that the results would support thefinding that addition of an XTEN to CD40 can greatly increase theterminal half-life compared to the targeting moiety not linked to XTEN,and enhance other pharmacokinetic parameters, as well.

Example 45: Preclinical Analysis of CTLA4-XTEN

CTLA4 is involved in delivery of the second co-stimulatory signalrequired for optimal activation of T cells. As such, the in vivopharmacologic activity of CTLA4-XTEN constructs can be assessed usingpreclinical models of human autoimmune inflammatory diseases.Appropriate models for preclinical efficacy testing include but are notlimited to collagen induced arthritis, a model for human rheumatoidarthritis, systemic lupus erythematosus, a model for human lupus, andexperimental allergic encephalomyelitis, a model for human multiplesclerosis. Preclinical efficacy testing can also be done intransplantation models such as solid organ allograft or islettransplant. These models can be developed in multiple species usingmethods equivalent to those used for abatacept. CTLA4-XTEN compositionsare provided in an aqueous buffer compatible with in vivo administration(for example: phosphate-buffered saline or Tris-buffered saline). Thecompositions would be administered at appropriate doses, dosingfrequency, dosing schedule and route of administration as optimized forthe particular model. Efficacy readouts for inflammation could includejoint measurements, inhibition of primary and secondary humoral immuneresponse, infiltration of immune cells as measured by histopathology,proteinuria among others. It is expected that the results would supportthe finding that the CTLA4-XTEN constructs may be more efficacious atinhibiting the inflammatory response as compared to CTLA4 and/orequivalent in potency to comparable dosage CTLA4 with less frequent ormore convenient dosing.

Example 46: Preclinical Analysis of Anti-IL6R-XTEN

IL6 plays an important role in the pathogenesis of rheumatoid arthritis.Anti-IL6R inhibits binding of IL6 to its receptor and neutralizes theactions of IL6. As such, the in vivo pharmacologic activity ofanti-IL6R-XTEN constructs can be assessed using preclinical models ofhuman autoimmune inflammatory diseases, in particular, rheumatoidarthritis. Appropriate models for preclinical efficacy testing includebut are not limited to non human primate collagen induced arthritis, amodel for human rheumatoid arthritis. These models can be developedusing methods equivalent to those used for tocilizumab. Anti-IL6R-XTENcompositions are provided in an aqueous buffer compatible with in vivoadministration (for example: phosphate-buffered saline or Tris-bufferedsaline). The compositions would be administered at appropriate doses,dosing frequency, dosing schedule and route of administration asoptimized for the particular model. Efficacy readouts for inflammationcould include joint measurements, swelling, inhibition of primary andsecondary humoral immune response, infiltration of immune cells asmeasured by histopathology, blood chemistry, among others. It isexpected that the results would support the finding that theanti-IL6R4-XTEN constructs may be more efficacious at inhibiting theinflammatory response as compared to anti-IL6R and/or equivalent inpotency to comparable dosage anti-IL6R with less frequent or moreconvenient dosing.

Example 47: Preclinical Analysis of Anti-CD40-XTEN

Dysregulation of the CD40-CD40L costimulation pathway plays an importantrole in the pathogenesis of human inflammatory and autoimmune disease.As such, the in vivo pharmacologic activity of anti-CD40-XTEN constructscan be assessed using preclinical models of human autoimmuneinflammatory diseases. Appropriate models for preclinical efficacytesting include but are not limited to collagen induced arthritis, amodel for human rheumatoid arthritis, systemic lupus erythematosus, amodel for human lupus, and experimental allergic encephalomyelitis, amodel for human multiple sclerosis. Preclinical efficacy testing canalso be done in transplantation models such as solid organ allograft orislet transplant. These models can be developed using methods equivalentto those used for other CD40 or CD40L targeting therapies.Anti-CD40-XTEN compositions can be provided in an aqueous buffercompatible with in vivo administration (for example phosphate-bufferedsaline or Tris-buffered saline). The compositions can be administered atappropriate doses, dosing frequency, dosing schedule and route ofadministration as optimized for the particular model. Efficacy readoutsfor inflammation could include joint measurements, inhibition of primaryand secondary humoral immune response, infiltration of immune cells asmeasured by histopathology, proteinuria among others. It is expectedthat the results would support the finding that the anti-CD40-XTENconstructs may be more efficacious at inhibiting the inflammatoryresponse as compared to anti-CD40 and/or equivalent in potency tocomparable dosage anti-CD40 with less frequent or more convenientdosing.

Example 48: Clinical Applications of CTLA4-XTEN

CTLA4 is involved in delivery of the second co-stimulatory signalrequired for optimal activation of T cells. CTLA4-XTEN can be used totreat T cell mediated autoimmune diseases such as rheumatoid arthritisand psoriasis in clinical trials using similar methodology to Orencia.Fusion of XTEN to CTLA4 to create a binding fusion protein compositionis expected to improve the half-life of the recombinant protein, thusenabling a lower overall dose per patient with subsequent improvementsin convenience (allowing for subcutaneous dosing, reducing dosingfrequency, etc) and cost (reduced drug required per dose).

Clinical trials could be conducted in patients suffering from rheumatoidarthritis. Clinical trials can be designed such that the efficacy andadvantages of the CTLA4-XTEN compositions can be verified in humans.Such studies in patients would comprise three phases. First, a Phase Isafety and pharmacokinetics study in adult patients would be conductedto determine the maximum tolerated dose and pharmacokinetics andpharmacodynamics in humans. These initial studies could be performed inpatients with rheumatoid arthritis and would define potential toxicitiesand adverse events to be tracked in future studies. The scheme of thestudy would be to use single escalating doses of CTLA4-XTEN compositionsand measure the biochemical, PK, and clinical parameters. This wouldpermit the determination of the maximum tolerated dose and establish thethreshold and maximum concentrations in dosage and circulating drug thatconstitute the therapeutic window to be used in subsequent Phase II andPhase III trials conducted in target indications to determine efficacyand tolerability of the CTLA4-XTEN compositions.

A phase II clinical study of human patients would be conducted inarthritis patients administered CTLA4-XTEN or a suitableanti-inflammatory protein to determine an appropriate dose to relieve atleast one symptom associated with rheumatoid arthritis, includingreducing joint swelling, joint tenderness, inflammation, morningstiffness, and pain, or at least one biological surrogate markerassociated with rheumatoid arthritis, including reducing erythrocytesedimentation rates, and serum levels of C-reactive protein and/or IL2receptor. In addition, safety data related to adverse events would becollected. A phase III efficacy study would be conducted whereinarthritis patients would be administered either the CTLA4-XTEN, apositive control, or a placebo daily, bi-weekly, or weekly (or otherdosing schedule deemed appropriate given the pharmacokinetic andpharmacodynamic properties of the compound) for an extended period oftime. Patients would be evaluated for baseline symptoms of diseaseactivity prior to receiving any treatments, including joint swelling,joint tenderness, inflammation, morning stiffness, disease activityevaluated by patient and physician as well as disability evaluated by,for example, a standardized Health Questionnaire Assessment (HAQ), andpain. Additional baseline evaluations could include erythrocytesedimentation rates (ESR), serum levels of C-reactive protein (CRP) andsoluble IL-2 receptor (IL-2r). The clinical response to treatment couldbe assessed using the criteria established by the American College ofRheumatology (ACR), such as the ACR20 criterion; i.e., if there was a 20percent improvement in tender and swollen joint counts and 20 percentimprovement in three of the five remaining symptoms measured, such aspatient and physician global disease changes, pain, disability, and anacute phase reactant (Felson, D. T., et al., 1993 Arthritis andRheumatism 36:729-740; Felson, D. T., et al., 1995 Arthritis andRheumatism 38:1-9). Similarly, a subject would satisfy the ACR50 orACR70 criterion if there was a 50 or 70 percent improvement,respectively, in tender and swollen joint counts and 50 or 70 percentimprovement, respectively, in three of the five remaining symptomsmeasured, such as patient and physician global disease changes, pain,physical disability, and an acute phase reactant such as CRP or ESR. Inaddition, potential biomarkers of disease activity could be measured,including rheumatoid factor, CRP, ESR, soluble IL-2R, soluble ICAM-1,soluble E-selectin, and MMP-3. Efficacy outcomes would be determinedusing standard statistical methods. Toxicity and adverse event markerswould also be followed in this study to verify that the compound is safewhen used in the manner described.

Example 49: Clinical Applications of IL6R-XTEN

IL6 plays an important role in the pathogenesis of rheumatoid arthritis.Anti-IL6R inhibits binding of IL6 to its receptor and neutralizes theactions of IL6. Anti-IL6R-XTEN can be used to treat T cell mediatedautoimmune diseases such as rheumatoid arthritis in clinical trialsusing similar methodology to Actemra. Fusion of XTEN to anti-IL6R tocreate a binding fusion protein is expected to improve the half-life ofthe recombinant protein, thus enabling a lower overall dose per patientwith subsequent improvements in convenience (allowing for subcutaneousdosing, reducing dosing frequency, etc) and cost (reduced drug requiredper dose). Anti-IL6R-XTEN may also provide a safety advantage over theexisting anti-IL6R therapy.

Clinical trials could be conducted in patients suffering from rheumatoidarthritis. Clinical trials can be designed such that the efficacy andadvantages of the anti-IL6R-XTEN compositions can be verified in humans.Such studies in patients would comprise three phases. First, a Phase Isafety and pharmacokinetics study in adult patients would be conductedto determine the maximum tolerated dose and pharmacokinetics andpharmacodynamics in humans. These initial studies could be performed inpatients with rheumatoid arthritis and would define potential toxicitiesand adverse events to be tracked in future studies. The scheme of thestudy would be to use single escalating doses of anti-IL6R-XTENcompositions and measure the biochemical, PK, and clinical parameters.This would permit the determination of the maximum tolerated dose andestablish the threshold and maximum concentrations in dosage andcirculating drug that constitute the therapeutic window to be used insubsequent Phase II and Phase III trials conducted in target indicationsto determine efficacy and tolerability of the anti-IL6R-XTENcompositions.

A phase II clinical study of human patients would be conducted inarthritis patients administered anti-IL6R-XTEN or a suitableanti-inflammatory protein to determine an appropriate dose to relieve atleast one symptom associated with rheumatoid arthritis, includingreducing joint swelling, joint tenderness, inflammation, morningstiffness, and pain, or at least one biological surrogate markerassociated with rheumatoid arthritis, including reducing erythrocytesedimentation rates, and serum levels of C-reactive protein and/or IL2receptor. In addition, safety data related to adverse events would becollected. A phase III efficacy study would be conducted whereinarthritis patients would be administered either the anti-IL6R-XTEN, apositive control, or a placebo daily, bi-weekly, or weekly (or otherdosing schedule deemed appropriate given the pharmacokinetic andpharmacodynamic properties of the compound) for an extended period oftime. Patients would be evaluated for baseline symptoms of diseaseactivity prior to receiving any treatments, including joint swelling,joint tenderness, inflammation, morning stiffness, disease activityevaluated by patient and physician as well as disability evaluated by,for example, a standardized Health Questionnaire Assessment (HAQ), andpain. Additional baseline evaluations could include erythrocytesedimentation rates (ESR), serum levels of C-reactive protein (CRP) andsoluble IL-2 receptor (IL-2r). The clinical response to treatment couldbe assessed using the criteria established by the American College ofRheumatology (ACR), such as the ACR20 criterion; i.e., if there was a 20percent improvement in tender and swollen joint counts and 20 percentimprovement in three of the five remaining symptoms measured, such aspatient and physician global disease changes, pain, disability, and anacute phase reactant (Felson, D. T., et al., 1993 Arthritis andRheumatism 36:729-740; Felson, D. T., et al., 1995 Arthritis andRheumatism 38:1-9). Similarly, a subject would satisfy the ACR50 orACR70 criterion if there was a 50 or 70 percent improvement,respectively, in tender and swollen joint counts and 50 or 70 percentimprovement, respectively, in three of the five remaining symptomsmeasured, such as patient and physician global disease changes, pain,physical disability, and an acute phase reactant such as CRP or ESR. Inaddition, potential biomarkers of disease activity could be measured,including rheumatoid factor, CRP, ESR, soluble IL-2R, soluble ICAM-1,soluble E-selectin, and MMP-3. Efficacy outcomes would be determinedusing standard statistical methods. Toxicity and adverse event markerswould also be followed in this study to verify that the compound is safewhen used in the manner described.

Example 50: Clinical Applications of Anti-CD40-XTEN

Dysregulation of the CD40-CD40L costimulation pathway plays an importantrole in the pathogenesis of human inflammatory and autoimmune disease.CD40 is over-expressed on antigen presenting cells in a variety ofautoimmune conditions including rheumatoid arthritis, psoriasis,inflammatory bowel disease, and type 1 diabetes, and its ligand, CD154,is over-expressed on T cells in many of these same autoimmune diseases.A binding fusion protein of anti-CD40-XTEN could be used to evaluateefficacy in autoimmune inflammatory diseases and its ability to inducetransplantation tolerance in clinical trials using similar methodologyto the anti-CD40 antibodies currently in clinical trials. Fusion of XTENto anti-CD40 to create a binding fusion protein is expected to improvethe half-life of the recombinant protein, thus enabling a lower overalldose per patient with subsequent improvements in convenience (reduceddosing frequency, etc) and cost (reduced drug required per dose).

Clinical trials could be conducted in patients suffering from any avariety of inflammatory and autoimmune conditions such as but notlimited to rheumatoid arthritis, lupus erythematosus, psoriasis,inflammatory bowel disease, multiple sclerosis, etc. or fortransplantation. Clinical trials can be designed such that the efficacyand advantages of the anti-CD40-XTEN compositions can be verified inhumans Such studies in patients would comprise three phases. First, aPhase I safety and pharmacokinetics study in adult patients would beconducted to determine the maximum tolerated dose and pharmacokineticsand pharmacodynamics in humans. These studies would define potentialtoxicities and adverse events to be tracked in future studies. Thescheme of the study would be to use single escalating doses ofanti-CD40-XTEN compositions and measure the biochemical, PK, andclinical parameters. This would permit the determination of the maximumtolerated dose and establish the threshold and maximum concentrations indosage and circulating drug that constitute the therapeutic window to beused in subsequent Phase II and Phase III trials conducted in targetindications to determine efficacy and tolerability of the anti-CD40-XTENcompositions.

A phase II clinical study of human patients would be conducted inarthritis patients administered anti-CD40-XTEN or a suitableanti-inflammatory protein to determine an appropriate dose to relieve atleast one symptom associated with rheumatoid arthritis, includingreducing joint swelling, joint tenderness, inflammation, morningstiffness, and pain, or at least one biological surrogate markerassociated with rheumatoid arthritis, including reducing erythrocytesedimentation rates, and serum levels of C-reactive protein and/or IL2receptor. In addition, safety data related to adverse events would becollected. A phase III efficacy study would be conducted whereinarthritis patients would be administered either the anti-CD40-XTEN, apositive control, or a placebo daily, bi-weekly, or weekly (or otherdosing schedule deemed appropriate given the pharmacokinetic andpharmacodynamic properties of the compound) for an extended period oftime. Patients would be evaluated for baseline symptoms of diseaseactivity prior to receiving any treatments, including joint swelling,joint tenderness, inflammation, morning stiffness, disease activityevaluated by patient and physician as well as disability evaluated by,for example, a standardized Health Questionnaire Assessment (HAQ), andpain. Additional baseline evaluations could include erythrocytesedimentation rates (ESR), serum levels of C-reactive protein (CRP) andsoluble IL-2 receptor (IL-2r). The clinical response to treatment couldbe assessed using the criteria established by the American College ofRheumatology (ACR), such as the ACR20 criterion; i.e., if there was a 20percent improvement in tender and swollen joint counts and 20 percentimprovement in three of the five remaining symptoms measured, such aspatient and physician global disease changes, pain, disability, and anacute phase reactant (Felson, D. T., et al., 1993 Arthritis andRheumatism 36:729-740; Felson, D. T., et al., 1995 Arthritis andRheumatism 38:1-9) Similarly, a subject would satisfy the ACR50 or ACR70criterion if there was a 50 or 70 percent improvement, respectively, intender and swollen joint counts and 50 or 70 percent improvement,respectively, in three of the five remaining symptoms measured, such aspatient and physician global disease changes, pain, physical disability,and an acute phase reactant such as CRP or ESR. In addition, potentialbiomarkers of disease activity could be measured, including rheumatoidfactor, CRP, ESR, soluble IL-2R, soluble ICAM-1, soluble E-selectin, andMMP-3. Efficacy outcomes would be determined using standard statisticalmethods. Toxicity and adverse event markers would also be followed inthis study to verify that the compound is safe when used in the mannerdescribed.

Example 51: Clinical Applications of aHER2-XTEN-aCD3

Her2 antigen is over-expressed on a large number of solid malignancies.Expression is particularly high on many breast cancer cells. Herceptinhas been approved for the treatment of HER2-positive breast cancers. Abinding fusion protein of anti-Her2-XTEN-anti-CD3 could be evaluated forefficacy in the treatment of the same patient population. Clinicaltrials can be designed such that the efficacy and advantages of theaHER2-XTEN-aCD3 compositions can be verified in humans. Such studies inpatients would comprise three phases. First, a Phase I safety andpharmacokinetics study in adult patients would be conducted to determinethe maximum tolerated dose and pharmacokinetics and pharmacodynamics inhumans. These studies would define potential toxicities and adverseevents to be tracked in future studies. The scheme of the study would beto use single escalating doses of aHER2-XTEN-aCD3 compositions andmeasure the biochemical, PK, and clinical parameters. This would permitthe determination of the maximum tolerated dose and establish thethreshold and maximum concentrations in dosage and circulating drug thatconstitute the therapeutic window to be used in subsequent Phase II andPhase III trials conducted in target indications to determine efficacyand tolerability of the aHER2-XTEN-aCD3 compositions.

Example 52: Characterization of Secondary Structure of Fusion ProteinComprising XTEN

A fusion protein consisting of the XTEN AE864 linked to a payload ofexenatide was evaluated for degree of secondary structure by circulardichroism spectroscopy. CD spectroscopy was performed on a Jasco J-715(Jasco Corporation, Tokyo, Japan) spectropolarimeter equipped with JascoPeltier temperature controller (TPC-348WI). The concentration of proteinwas adjusted to 0.2 mg/mL in 20 mM sodium phosphate pH 7.0, 50 mM NaCl.The experiments were carried out using HELLMA quartz cells with anoptical path-length of 0.1 cm. The CD spectra were acquired at 5°, 25°,45°, and 65° C. and processed using the J-700 version 1.08.01 (Build 1)Jasco software for Windows. The samples were equilibrated at eachtemperature for 5 min before performing CD measurements. All spectrawere recorded in duplicate from 300 nm to 185 nm using a bandwidth of 1nm and a time constant of 2 sec, at a scan speed of 100 nm/min. The CDspectrum shown in FIG. 30 shows no evidence of stable secondarystructure and is consistent with an unstructured polypeptide.

Example 53: Pharmacokinetics of Extended Polypeptides Fused to GFP inCynomolgus Monkeys

The pharmacokinetics of GFP-L288, GFP-L576, GFP-XTEN_AF576,GFP-XTEN_Y576 and XTEN_AD836-GFP were tested in cynomolgus monkeys todetermine the effect of composition and length of the unstructuredpolypeptides on PK parameters. Blood samples were analyzed at varioustimes after injection and the concentration of GFP in plasma wasmeasured by ELISA using a polyclonal antibody against GFP for captureand a biotinylated preparation of the same polyclonal antibody fordetection. Results are summarized in FIG. 31. They show a surprisingincrease of half-life with increasing length of the XTEN sequence. Forexample, a half-life of 10 h was determined for GFP-XTEN_L288 (with 288amino acid residues in the XTEN). Doubling the length of theunstructured polypeptide fusion partner to 576 amino acids increased thehalf-life to 20-22 h for multiple fusion protein constructs; i.e.,GFP-XTEN_L576, GFP-XTEN_AF576, GFP-XTEN_Y576. A further increase of theunstructured polypeptide fusion partner length to 836 residues resultedin a half-life of 72-75 h for XTEN_AD836-GFP. Thus, increasing thepolymer length by 288 residues from 288 to 576 residues increased invivo half-life by about 10 h. However, increasing the polypeptide lengthby 260 residues from 576 residues to 836 residues increased half-life bymore than 50 h. These results show that there is a surprising thresholdof unstructured polypeptide length that results in a greater thanproportional gain in in vivo half-life. Thus, fusion proteins comprisingextended, unstructured polypeptides are expected to have the property ofenhanced pharmacokinetics compared to polypeptides of shorter lengths.

Example 54: Increasing Solubility and Stability of a Peptide Payload byLinking to XTEN

In order to evaluate the ability of XTEN to enhance the physicochemicalproperties of solubility and stability, fusion proteins of glucagon plusshorter-length XTEN were prepared and evaluated. The test articles wereprepared in Tris-buffered saline at neutral pH and characterization ofthe Gcg-XTEN solution was by reverse-phase HPLC and size exclusionchromatography to affirm that the protein was homogeneous andnon-aggregated in solution. The data are presented in Table 29. Forcomparative purposes, the solubility limit of unmodified glucagon in thesame buffer was measured at 60 μM (0.2 mg/mL), and the resultdemonstrate that for all lengths of XTEN added, a substantial increasein solubility was attained. Importantly, in most cases the glucagon-XTENfusion proteins were prepared to achieve target concentrations and werenot evaluated to determine the maximum solubility limits for the givenconstruct. However, in the case of glucagon linked to the AF-144 XTEN,the limit of solubility was determined, with the result that a 60-foldincrease in solubility was achieved, compared to glucagon not linked toXTEN. In addition, the glucagon-AF144 CFXTEN was evaluated forstability, and was found to be stable in liquid formulation for at least6 months under refrigerated conditions and for approximately one monthat 37° C. (data not shown).

The data support the conclusion that the linking of short-length XTENpolypeptides to a biologically active protein such as glucagon canmarkedly enhance the solubility properties of the protein by theresulting fusion protein, as well as confer stability at the higherprotein concentrations.

TABLE 29 Solubility of Glucagon-XTEN constructs Test Article SolubilityGlucagon 60 μM Glucagon-Y36 >370 μM Glucagon-Y72 >293 μMGlucagon-AF108 >145 μM Glucagon-AF120 >160 μM Glucagon-Y144 >497 μMGlucagon-AE144 >467 μM Glucagon-AF144 >3600 μM Glucagon-Y288 >163 μM

Example 55: Binding Fusion Proteins with Cleavage Sequences

C-Terminal XTEN Releasable by FXIa

A fusion protein consisting of an XTEN protein fused to the C-terminusof a targeting moiety can be created with a XTEN release site cleavagesequence placed in between the targeting moiety and XTEN components. Inthis case, the release site cleavage sequence can be incorporated intothe XTEN that contains an amino acid sequence that is recognized andcleaved by the FXIa protease (EC 3.4.21.27, Uniprot P03951).Specifically the amino acid sequence KLTRAET (SEQ ID NO: 748) is cutafter the arginine of the sequence by FXIa protease. FXI is thepro-coagulant protease located immediately before FVIII in the intrinsicor contact activated coagulation pathway. Active FXIa is produced fromFXI by proteolytic cleavage of the zymogen by FXIIa. Production of FXIais tightly controlled and only occurs when coagulation is necessary forproper hemostasis. Therefore, by incorporation of the KLTRAET (SEQ IDNO: 748) cleavage sequence, the XTEN domain is removed from targetingmoiety concurrent with activation of the intrinsic coagulation pathwayin proximity to the targeting moiety-XTEN. This creates a situationwhere the targeting moiety-XTEN fusion protein is processed in oneadditional manner during the activation of the intrinsic pathway.

C-Terminal XTEN Releasable by Elastase-2

A fusion protein consisting of an XTEN protein fused to the C-terminusof a targeting moiety can be created with a XTEN release site cleavagesequence placed in between the targeting moiety and XTEN components. Inthis case, the release site contains an amino acid sequence that isrecognized and cleaved by the elastase-2 protease (EC 3.4.21.37, UniprotP08246). Specifically the sequence LGPVSGVP (SEQ ID NO: 749) [RawlingsN. D., et al. (2008) Nucleic Acids Res., 36: D320], is cut afterposition 4 in the sequence. Elastase is constitutively expressed byneutrophils and is present at all times in the circulation, butparticularly during acute inflammation. Therefore as the long livedtargeting moiety-XTEN circulates, a fraction of it is cleaved,particularly locally during inflammatory responses, creating a pool ofshorter-lived targeting moiety to be used at the site of inflammation.

C-Terminal XTEN Releasable by MMP-12

A fusion protein consisting of an XTEN protein fused to the C-terminusof a targeting moiety can be created with a XTEN release site cleavagesequence placed in between the targeting moiety and XTEN components. Inthis case, the release site contains an amino acid sequence that isrecognized and cleaved by the MMP-12 protease (EC 3.4.24.65, UniprotP39900). Specifically the sequence GPAGLGGA (SEQ ID NO: 750) [RawlingsN. D., et al. (2008) Nucleic Acids Res., 36: D320], is cut afterposition 4 of the sequence. MMP-12 is constitutively expressed in wholeblood. Therefore as the long lived AAT-XTEN circulates, a fraction of itis cleaved, creating a pool of shorter-lived AAT to be used. In adesirable feature of the inventive composition, this creates acirculating pro-drug depot that constantly releases a prophylacticamount of targeting moiety, with higher amounts released during aninflammatory response.

C-Terminal XTEN Releasable by MMP-13

A fusion protein consisting of an XTEN protein fused to the C-terminusof a targeting moiety can be created with a XTEN release site cleavagesequence placed in between the targeting moiety and XTEN components. Inthis case, the release site contains an amino acid sequence that isrecognized and cleaved by the MMP-13 protease (EC 3.4.24.-, UniprotP45452). Specifically the sequence GPAGLRGA (SEQ ID NO: 751) [RawlingsN. D., et al. (2008) Nucleic Acids Res., 36: D320], is cut afterposition 4. MMP-13 is constitutively expressed in whole blood. Thereforeas the long lived targeting moiety-XTEN circulates, a fraction of it iscleaved, creating a pool of shorter-lived AAT to be used. In a desirablefeature of the inventive composition, this creates a circulatingpro-drug depot that constantly releases a prophylactic amount oftargeting moiety, with higher amounts released during an inflammatoryresponse.

C-Terminal XTEN Releasable by MMP-17

A fusion protein consisting of an XTEN protein fused to the C-terminusof a targeting moiety can be created with a XTEN release site cleavagesequence placed in between the targeting moiety and XTEN components. Inthis case, the release site contains an amino acid sequence that isrecognized and cleaved by the MMP-20 protease (EC.3.4.24.-, UniprotQ9ULZ9). Specifically the sequence APLGLRLR (SEQ ID NO: 752) [RawlingsN. D., et al. (2008) Nucleic Acids Res., 36: D320], is cut afterposition 4 in the sequence. MMP-17 is constitutively expressed in wholeblood. Therefore as the long lived targeting moiety-XTEN circulates, afraction of it is cleaved, creating a pool of shorter-lived targetingmoiety to be used. In a desirable feature of the inventive composition,this creates a circulating pro-drug depot that constantly releases aprophylactic amount of targeting moiety, with higher amounts releasedduring an inflammatory response.

C-Terminal XTEN Releasable by MMP-20

A fusion protein consisting of an XTEN protein fused to the C-terminusof a targeting moiety can be created with a XTEN release site cleavagesequence placed in between the targeting moiety and XTEN components. Inthis case, the release site contains an amino acid sequence that isrecognized and cleaved by the MMP-20 protease (EC.3.4.24.-, Uniprot060882). Specifically the sequence PALPLVAQ (SEQ ID NO: 753) [RawlingsN. D., et al. (2008) Nucleic Acids Res., 36: D320], is cut afterposition 4 (depicted by the arrow). MMP-20 is constitutively expressedin whole blood. Therefore as the long lived targeting moiety-XTENcirculates, a fraction of it is cleaved, creating a pool ofshorter-lived targeting moiety to be used. In a desirable feature of theinventive composition, this creates a circulating pro-drug depot thatconstantly releases a prophylactic amount of targeting moiety, withhigher amounts released during an inflammatory response.

Example 56: Serum Stability of XTEN

A fusion protein containing XTEN_AE864 fused to the N-terminus of GFPwas incubated in monkey plasma and rat kidney lysate for up to 7 days at37° C. Samples were withdrawn at time 0, Day 1 and Day 7 and analyzed bySDS PAGE followed by detection using Western analysis and detection withantibodies against GFP as shown in FIG. 16A-FIG. 16C. The sequence ofXTEN_AE864 showed negligible signs of degradation over 7 days in plasma.However, XTEN_AE864 was rapidly degraded in rat kidney lysate over 3days. The in vivo stability of the fusion protein was tested in plasmasamples wherein the GFP_AE864 was immunoprecipitated and analyzed by SDSPAGE as described above. Samples that were withdrawn up to 7 days afterinjection showed very few signs of degradation. The results demonstratethe resistance of binding fusion protein to degradation due to serumproteases; a factor in the enhancement of pharmacokinetic properties ofthe binding fusion proteins.

Example 57: Analysis of Sequences for Secondary Structure by PredictionAlgorithms

Amino acid sequences can be assessed for secondary structure via certaincomputer programs or algorithms, such as the well-known Chou-Fasmanalgorithm (Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45) and theGarnier-Osguthorpe-Robson, or “GOR” method (Gamier J, Gibrat J F, RobsonB. (1996). GOR method for predicting protein secondary structure fromamino acid sequence. Methods Enzymol 266:540-553). For a given sequence,the algorithms can predict whether there exists some or no secondarystructure at all, expressed as total and/or percentage of residues ofthe sequence that form, for example, alpha-helices or beta-sheets or thepercentage of residues of the sequence predicted to result in randomcoil formation.

Several representative sequences from XTEN “families” have been assessedusing two algorithm tools for the Chou-Fasman and GOR methods to assessthe degree of secondary structure in these sequences. The Chou-Fasmantool was provided by William R Pearson and the University of Virginia,at the “Biosupport” internet site, URL located on the World Wide Web atfasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=miscl as it existedon Jun. 19, 2009. The GOR tool was provided by Pole InformatiqueLyonnais at the Network Protein Sequence Analysis internet site, URLlocated on the World Wide Web at.npsa-pbil.ibcp.fr/cgi-bin/secpred_gor4.p1 as it existed on Jun. 19,2008.

As a first step in the analyses, a single XTEN sequence was analyzed bythe two algorithms. The AE864 composition is a XTEN with 864 amino acidresidues created from multiple copies of four 12 amino acid sequencemotifs consisting of the amino acids G, S, T, E, P, and A. The sequencemotifs are characterized by the fact that there is limitedrepetitiveness within the motifs and within the overall sequence in thatthe sequence of any two consecutive amino acids is not repeated morethan twice in any one 12 amino acid motif, and that no three contiguousamino acids of full-length the XTEN are identical. Successively longerportions of the AF 864 sequence from the N-terminus were analyzed by theChou-Fasman and GOR algorithms (the latter requires a minimum length of17 amino acids). The sequences were analyzed by entering the FASTAformat sequences into the prediction tools and running the analysis. Theresults from the analyses are presented in Table 30.

The results indicate that, by the Chou-Fasman calculations, short XTENof the AE and AG families, up to at least 288 amino acid residues, haveno alpha-helices or beta sheets, but amounts of predicted percentage ofrandom coil by the GOR algorithm vary from 78-99%. With increasing XTENlengths of 504 residues to greater than 1300, the XTEN analyzed by theChou-Fasman algorithm had predicted percentages of alpha-helices or betasheets of 0 to about 2%, while the calculated percentages of random coilincreased to from 94-99%. Those XTEN with alpha-helices or beta sheetswere those sequences with one or more instances of three contiguousserine residues, which resulted in predicted beta-sheet formation.However, even these sequences still had approximately 99% random coilformation.

The analysis supports the conclusion that: 1) XTEN created from multiplesequence motifs of G, S, T, E, P, and A that have limited repetitivenessas to contiguous amino acids are predicted to have very low amounts ofalpha-helices and beta-sheets; 2) that increasing the length of the XTENdoes not appreciably increase the probability of alpha-helix orbeta-sheet formation; and 3) that progressively increasing the length ofthe XTEN sequence by addition of non-repetitive 12-mers consisting ofthe amino acids G, S, T, E, P, and A results in increased percentage ofrandom coil formation. Based on the numerous sequences evaluated bythese methods, it is concluded that XTEN created from sequence motifs ofG, S, T, E, P, and A that have limited repetitiveness (defined as nomore than two identical contiguous amino acids in any one motif) areexpected to have very limited secondary structure. With the exception ofmotifs containing three contiguous serines, it is believed that anyorder or combination of sequence motifs from Table 3 can be used tocreate an XTEN polypeptide that will result in an XTEN sequence that issubstantially devoid of secondary structure, and that the effects ofthree contiguous serines is ameliorated by increasing the length of theXTEN. Such sequences are expected to have the characteristics describedin the CFXTEN embodiments of the invention disclosed herein.

TABLE 30CHOU-FASMAN and GOR prediction calculations of polypeptide sequences SEQSEQ ID No. Chou-Fasman GOR NAME Sequence NO: Residues CalculationCalculation AE36: GSPAGSPTSTEEGTSESATPES 754   36Residue totals: H: 0 E: 0 94.44% LCW0402_ GPGTSTEPSEGSAPpercent: H: 0.0 E: 0.0 002 AE36: GTSTEPSEGSAPGTSTEPSEGS 755   36Residue totals: H: 0 E: 0 94.44% LCW0402_ APGTSTEPSEGSAPpercent: H: 0.0 E: 0.0 003 AG36: GASPGTSSTGSPGTPGSGTASS 756   36Residue totals: H: 0 E: 0 77.78% LCW0404_ SPGSSTPSGATGSPpercent: H: 0.0 E: 0.0 001 AG36: GSSTPSGATGSPGSSPSASTGT 757   36Residue totals: H: 0 E: 0 83.33% LCW0404_ GPGSSTPSGATGSPpercent: H: 0.0 E: 0.0 003 AE42_1 TEPSEGSAPGSPAGSPTSTEEG 758   42Residue totals: H: 0 E: 0 90.48% TSESATPESGPGSEPATSGSpercent: H: 0.0 E: 0.0 AE42_1 TEPSEGSAPGSPAGSPTSTEEG 759   42Residue totals: H: 0 E: 0 90.48% TSESATPESGPGSEPATSGSpercent: H: 0.0 E: 0.0 AG42_1 GAPSPSASTGTGPGTPGSGTAS 760   42Residue totals: H: 0 E: 0 88.10% SSPGSSTPSGATGSPGPSGPpercent: H: 0.0 E: 0.0 AG42_2 GPGTPGSGTASSSPGSSTPSGA 761   42Residue totals: H: 0 E: 0 88.10% TGSPGSSPSASTGTGPGASPpercent: H: 0.0 E: 0.0 AE144 GSEPATSGSETPGTSESATPES 762  144Residue totals: H: 0 E: 0 98.61% GPGSEPATSGSETPGSPAGSPTpercent: H: 0.0 E: 0.0 STEEGTSTEPSEGSAPGSEPATS GSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGTS TEPSEGSAP AG144_1PGSSPSASTGTGPGSSPSASTGT 763  144 Residue totals: H: 0 E: 0 91.67%GPGTPGSGTASSSPGSSTPSGA percent: H: 0.0 E: 0.0 TGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP SGATGSPGTPGSGTASSSPGAS PGTSSTGSPGASPGTSSTGSPGTPGSGTASSS AE288 GTSESATPESGPGSEPATSGSE 764  288Residue totals: H: 0 E: 0 99.31% TPGTSESATPESGPGSEPATSGSpercent: H: 0.0 E: 0.0 ETPGTSESATPESGPGTSTEPSE GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSPAGSPTSTEEGSP AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP GTSESATPESGPGSEPATSGSE TPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPAG288_2 GSSPSASTGTGPGSSPSASTGT 765  288 Residue totals: H: 0 E: 0 92.71GPGTPGSGTASSSPGSSTPSGA percent: H: 0.0 E: 0.0 TGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP SGATGSPGTPGSGTASSSPGAS PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP GASPGTSSTGSPGTPGSGTASS SPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSG ATGSPGSSTPSGATGSPGASPG TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP AF504 GASPGTSSTGSPGSSPSASTGT 766  504Residue totals: H: 0 E: 0 94.44% GPGSSPSASTGTGPGTPGSGTApercent: H: 0.0 E: 0.0 SSSPGSSTPSGATGSPGSNPSAS TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTP GSGTASSSPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTG SPGTPGSGTASSSPGSSTPSGAT GSPGSNPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTP SGATGSPGASPGTSSTGSPGAS PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSP GASPGTSSTGSPGASPGTSSTG SPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTS STGSPGASPGTSSTGSPGSSTPS GATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP AD576 GSSESGSSEGGPGSGGEPSESG 767  576Residue totals: H: 7 E: 0 99.65% SSGSSESGSSEGGPGSSESGSSEpercent: H: 1.2 E: 0.0 GGPGSSESGSSEGGPGSSESGS SEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSE SGSSEGGPGSSESGSSEGGPGS SESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGS ESGSGGEPSESGSSGSSESGSSE GGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPG GSSGSESGSGGEPSESGSSGSG GEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSES GESPGGSSGSESGESPGGSSGS ESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEP SESGSSGSEGSSGPGESSGSSES GSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSG ESPGGSSGSESGESPGGSSGSES GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSES GSSGSGGEPSESGSSGESPGGS SGSESGSEGSSGPGESSGSSESGSSEGGPGSEGSSGPGESS AE576 GSPAGSPTSTEEGTSESATPES 768  576Residue totals: H: 2 E: 0 99.65% GPGTSTEPSEGSAPGSPAGSPTpercent: H: 0.4 E: 0.0 STEEGTSTEPSEGSAPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA GSPTSTEEGTSESATPESGPGTS TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPES GPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS EGSAPGTSTEPSEGSAPGTSES ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPS EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPES GPGSPAGSPTSTEEGSPAGSPT STEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AG576 PGTPGSGTASSSPGSSTPSGAT 769  576Residue totals: H: 0 E: 3 99.31% GSPGSSPSASTGTGPGSSPSASTpercent: H: 0.4 E: 0.5 GTGPGSSTPSGATGSPGSSTPS GATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGT PGSGTASSSPGASPGTSSTGSP GASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTA SSSPGASPGTSSTGSPGASPGTS STGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASP GTSSTGSPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP GASPGTSSTGSPGASPGTSSTG SPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS STGSPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG TPGSGTASSSPGSSTPSGATGSP GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSS TGSPGTPGSGTASSSPGSSTPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS AF540 GSTSSTAESPGPGSTSSTAESPG 770  540Residue totals: H: 2 E: 0 99.65 PGSTSESPSGTAPGSTSSTAESPpercent: H: 0.4 E: 0.0 GPGSTSSTAESPGPGTSTPESGS ASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESP SGTAPGTSPSGESSTAPGSTSES PSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTS ESPSGTAPGSTSESPSGTAPGTS TPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPG STSSTAESPGPGTSTPESGSASP GTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSA SPGSTSESPSGTAPGSTSESPSG TAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPES GSASPGSTSESPSGTAPGSTSES PSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTST PESGSASPGTSPSGESSTAPGST SSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPG STSESPSGTAP AD836 GSSESGSSEGGPGSSESGSSEG 771 836 Residue totals: H: 0 E: 0 98.44% GPGESPGGSSGSESGSGGEPSEpercent: H: 0.0 E: 0.0 SGSSGESPGGSSGSESGESPGG SSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGES PGGSSGSESGESPGGSSGSESG ESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEG GPGSSESGSSEGGPGSSESGSSE GGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPG GSSGSESGSGGEPSESGSSGSE GSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESS GSSESGSSEGGPGSGGEPSESG SSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSSESGSS EGGPGSGGEPSESGSSGSGGEP SESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGESSGSE GSSGPGESSGSGGEPSESGSSG SSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESG SSGSEGSSGPGESSGESPGGSS GSESGSEGSSGPGSSESGSSEGGPGSGGEPSESGSSGSEGSSGP GESSGSEGSSGPGESSGSEGSS GPGESSGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGES PGGSSGSESGSGGEPSESGSSG SEGSSGPGESSGESPGGSSGSESGSSESGSSEGGPGSSESGSSEG GPGSSESGSSEGGPGSGGEPSE SGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSSES GSSEGGPGESPGGSSGSESGSG GEPSESGSSGESPGGSSGSESGSGGEPSESGSS AE864 GSPAGSPTSTEEGTSESATPES 772  864Residue totals: H: 2 E: 3 99.77% GPGTSTEPSEGSAPGSPAGSPTpercent: H: 0.2 E: 0.4 STEEGTSTEPSEGSAPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA GSPTSTEEGTSESATPESGPGTS TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPES GPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS EGSAPGTSTEPSEGSAPGTSES ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPS EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPES GPGSPAGSPTSTEEGSPAGSPT STEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGS PAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT STEEGTSTEPSEGSAPGTSESAT PESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPA TSGSETPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPG TSTEPSEGSAP AF864 GSTSESPSGTAPGTSPSGESSTA 773 875 Residue totals: H: 2 E: 0 95.20% PGSTSESPSGTAPGSTSESPSGTpercent: H: 0.2 E: 0.0 APGTSTPESGSASPGTSTPESGS ASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESP SGTAPGTSPSGESSTAPGTSPS GESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGST SSTAESPGPGTSTPESGSASPGT STPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASP GSTSSTAESPGPGTSTPESGSAS PGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESS TAPGTSTPESGSASPGSTSSTAE SPGPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSG ESSTAPGSTSESPSGTAPGSTSE SPSGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGS TSESPSGTAPGSTSESPSGTAPG STSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSAS PGTSPSGESSTAPGTSPSGESST APGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPS GTAPGSTSESPSGTAPGTSPSG ESSTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTS ESPSGTAPGTSTPESGSASPGST SSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPG STSSTAESPGPGTSPSGESSTAP GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESST APGSTSSTAESPGPGSTSSTAES PGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPS GATGSP AG864 GASPGTSSTGSPGSSPSASTGT 774  864Residue totals: H: 0 E: 0 94.91% GPGSSPSASTGTGPGTPGSGTApercent: H: 0.0 E: 0.0 SSSPGSSTPSGATGSPGSSPSAS TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTP GSGTASSSPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTG SPGTPGSGTASSSPGSSTPSGAT GSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPS GATGSPGASPGTSSTGSPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSP GASPGTSSTGSPGASPGTSSTG SPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTS STGSPGASPGTSSTGSPGSSTPS GATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTG SPGTPGSGTASSSPGASPGTSST GSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGTP GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSP GSSTPSGATGSPGSSPSASTGT GPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG ATGSPGSSPSASTGTGPGSSPS ASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGS SPSASTGTGPGASPGTSSTGSP GSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT GSPGASPGTSSTGSP AM875 GTSTEPSEGSAPGSEPATSGSE 775 875 Residue totals: H: 7 E: 3 98.63% TPGSPAGSPTSTEEGSTSSTAESpercent: H: 0.8 E: 0.3 PGPGTSTPESGSASPGSTSESPS GTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPA TSGSETPGTSESATPESGPGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG TSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGTSTEPSEGSAPGTSTEPS EGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSP AGSPTSTEEGSSTPSGATGSPG TPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGSPAGSPT STEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESA TPESGPGSPAGSPTSTEEGSPA GSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGT PGSGTASSSPGSSTPSGATGSP GSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGS ETPGSTSSTAESPGPGSTSSTAE SPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEP SEGSAPGSTSSTAESPGPGTSTP ESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGSSTPSGATGSPG SSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGSSTPSGA TGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTE PSEGSAPGTSTEPSEGSAP AM1318GTSTEPSEGSAPGSEPATSGSE 776 1318 Residue totals: H: 7 E: 0 99.17%TPGSPAGSPTSTEEGSTSSTAES percent: H: 0.7 E: 0.0 PGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPES GSASPGTSTPESGSASPGSEPA TSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPG TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS APGTSESATPESGPGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTE PSEGSAPGSEPATSGSETPGSP AGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSP GTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS EGSAPGPEPTGPAPSGGSEPAT SGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSP AGSPTSTEEGSPAGSPTSTEEG TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPG PGSTSESPSGTAPGTSPSGESST APGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSE GSAPGTSESATPESGPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPG TSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTA PGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSG ATGSPGSSTPSGATGSPGSSTPS GATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGST SSTAESPGPGTSPSGESSTAPGT SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGP GSSTPSGATGSPGASPGTSSTG SPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATP ESGPGSEPATSGSETPGTSTEPS EGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPA GSPTSTEEGTSESATPESGPGTS TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GSSTPSGATGSPGASPGTSSTG SPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAE SPGPGSSTPSGATGSPGASPGT SSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTS TEPSEGSAP AM923 MAEPAGSPTSTEEGASPGTSST 777  924Residue totals: H: 4 E: 3 98.70% GSPGSSTPSGATGSPGSSTPSGpercent: H: 0.4 E: 0.3 ATGSPGTSTEPSEGSAPGSEPA TSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGST SESPSGTAPGSTSESPSGTAPGT STPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGP GSPAGSPTSTEEGTSTEPSEGS APGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS PTSTEEGTSTEPSEGSAPGTSTE PSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPG TSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATG SPGTPGSGTASSSPGSSTPSGAT GSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGS PTSTEEGSPAGSPTSTEEGTSTE PSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSP AGSPTSTEEGSTSSTAESPGPGS TSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSP GSSPSASTGTGPGSEPATSGSE TPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAE SPGPGTSPSGESSTAPGSEPATS GSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTP ESGSASPGSTSESPSGTAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPG SSPSASTGTGPGASPGTSSTGSP GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGA TGSPGSSPSASTGTGPGASPGT SSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP AE912 MAEPAGSPTSTEEGTPGSGTAS 778  913Residue totals: H: 8 E: 3 99.45% SSPGSSTPSGATGSPGASPGTSSpercent: H: 0.9 E: 0.3 TGSPGSPAGSPTSTEEGTSESA TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPGS EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES GPGTSTEPSEGSAPGTSTEPSE GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGTSTEPSEGSAPGTS ESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSESATPES GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPGTSTE PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGSPAGSPTSTEEG TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGSPAGSPTSTEEGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSEPATSGSE TPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESA TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPG TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTE EGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP BC864 GTSTEPSEPGSAGTSTEPSEPGS 779Residue totals: H: 0 E: 0 99.77% AGSEPATSGTEPSGSGASEPTSpercent: H: 0 E: 0 TEPGSEPATSGTEPSGSEPATS GTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEP ATSGTEPSGTSTEPSEPGSAGS EPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGS AGSEPATSGTEPSGSEPATSGT EPSGTSEPSTSEPGAGSGASEPTSTEPGTSEPSTSEPGAGSEPATS GTEPSGSEPATSGTEPSGTSTEP SEPGSAGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSE PATSGTEPSGSEPATSGTEPSGS EPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTST EPGTSTEPSEPGSAGSEPATSG TEPSGSGASEPTSTEPGTSTEPSEPGSAGSGASEPTSTEPGSEPA TSGTEPSGSGASEPTSTEPGSEP ATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSG SGASEPTSTEPGTSTEPSEPGSA GSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEP GSAGTSTEPSEPGSAGTSTEPS EPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSE PSTSEPGAGSGASEPTSTEPGTS TEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPS GSGASEPTSTEPGSEPATSGTE PSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSEPSTS EPGAGSEPATSGTEPSGSGASE PTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTST EPSEPGSA *H: alpha-helix E: beta-sheet

Example 58: Analysis of Polypeptide Sequences for Repetitiveness

Polypeptide amino acid sequences can be assessed for repetitiveness byquantifying the number of times a shorter subsequence appears within theoverall polypeptide. For example, a polypeptide of 200 amino acidresidues has 192 overlapping 9-amino acid subsequences (or 9-mer“frames”), but the number of unique 9-mer subsequences will depend onthe amount of repetitiveness within the sequence. In the presentanalysis, different sequences were assessed for repetitiveness bysumming the occurrence of all unique 3-mer subsequences for each 3-aminoacid frame across the first 200 amino acids of the polymer portiondivided by the absolute number of unique 3-mer subsequences within the200 amino acid sequence. The resulting subsequence score is a reflectionof the degree of repetitiveness within the polypeptide. The sequences ofTable 31 were analyzed by the algorithm SegScore (FIG. 37), whichapplies Equation I to the first 200 amino acids of a polypeptide. Theresults, shown in Table 31, indicate that the unstructured polypeptidesconsisting of 2 or 3 amino acid types have high subsequence scores,while those of consisting of 12 amino acids motifs of the six aminoacids G, S, T, E, P, and A with a low degree of internal repetitiveness,have subsequence scores of less than 10, and in some cases, less than 5.For example, the L288 sequence has two amino acid types and has short,highly repetitive sequences, resulting in a subsequence score of 50.0.The polypeptide J288 has three amino acid types but also has short,repetitive sequences, resulting in a subsequence score of 33.3. Y576also has three amino acid types, but is not made of internal repeats,reflected in the subsequence score of 15.7 over the first 200 aminoacids. W576 consists of four types of amino acids, but has a higherdegree of internal repetitiveness, e.g., “GGSG” (SEQ ID NO: 780),resulting in a subsequence score of 23.4. The AD576 consists of fourtypes of 12 amino acid motifs, each consisting of four types of aminoacids. Because of the low degree of internal repetitiveness of theindividual motifs, the overall subsequence score over the first 200amino acids is 13.6. In contrast, XTEN's consisting of four motifscontains six types of amino acids, each with a low degree of internalrepetitiveness have lower subsequence scores; i.e., AE864 (6.1), AF864(7.5), and AM875 (4.5).

Conclusions:

The results indicate that the combination of 12 amino acid subsequencemotifs, each consisting of four to six amino acid types that areessentially non-repetitive, into a longer XTEN polypeptide results in anoverall sequence that is non-repetitive. This is despite the fact thateach subsequence motif may be used multiple times across the sequence.In contrast, polymers created from smaller numbers of amino acid typesresulted in higher subsequence scores, although the actual sequence canbe tailored to reduce the degree of repetitiveness to result in lowersubsequence scores.

TABLE 31 Subsequence score calculations of polypeptide sequences SeqSEQ ID Name Amino Acid Sequence NO: Score J288GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG 781 33.3GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG K288GEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGE 782 46.9GGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEG L288SSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSES 78350.0 SESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSES Y288GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEGE 784 26.8GSGEGSEGEGGSEGSEGEGSGEGSEGEGSEGGSEGEGGSEGSEGEGSGEGSEGEGGEGGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGEGSEGSGE GEGSEGSGE Q576GGKPGEGGKPEGGGGKPGGKPEGEGEGKPGGKPEGGGKPGGGEGGKPEGGKPEG 785 18.5EGKPGGGEGKPGGKPEGGGGKPEGEGKPGGGGGKPGGKPEGEGKPGGGEGGKPEGKPGEGGEGKPGGKPEGGGEGKPGGGKPGEGGKPGEGKPGGGEGGKPEGGKPEGEGKPGGGEGKPGGKPGEGGKPEGGGEGKPGGKPGEGGEGKPGGGKPEGEGKPGGGKPGGGEGGKPEGEGKPGGKPEGGGEGKPGGKPEGGGKPEGGGEGKPGGGKPGEGGKPGEGEGKPGGKPEGEGKPGGEGGGKPEGKPGGGEGGKPEGGKPGEGGKPEGGKPGEGGEGKPGGGKPGEGGKPEGGGKPEGEGKPGGGGKPGEGGKPEGGKPEGGGEGKPGGGKPEGEGKPGGGEGKPGGKPEGGGGKPGEGGKPEGGKPGGEGGGKPEGEGKPGGKPGEGGGGKPGGKPEGEGKPGEGGEGKPGGKPEGGGEGKPGGKPEGGGEGKPGGGKPGEGGKPEGGGKPGEGGKPGEGGKPEGEGKPGGGEGKPGGKPGEGGKPEGGGEGKPGGKPGGEGGGKPEGGKPGEGGKPEG U576GEGKPGGKPGSGGGKPGEGGKPGSGEGKPGGKPGSGGSGKPGGKPGEGGKPEGG 786 18.1SGGKPGGGGKPGGKPGGEGSGKPGGKPEGGGKPEGGSGGKPGGKPEGGSGGKPGGKPGSGEGGKPGGGKPGGEGKPGSGKPGGEGSGKPGGKPEGGSGGKPGGKPEGGSGGKPGGSGKPGGKPGEGGKPEGGSGGKPGGSGKPGGKPEGGGSGKPGGKPGEGGKPGSGEGGKPGGGKPGGEGKPGSGKPGGEGSGKPGGKPGSGGEGKPGGKPEGGSGGKPGGGKPGGEGKPGSGGKPGEGGKPGSGGGKPGGKPGGEGEGKPGGKPGEGGKPGGEGSGKPGGGGKPGGKPGGEGGKPEGSGKPGGGSGKPGGKPEGGGGKPEGSGKPGGGGKPEGSGKPGGGKPEGGSGGKPGGSGKPGGKPGEGGGKPEGSGKPGGGSGKPGGKPEGGGKPEGGSGGKPGGKPEGGSGGKPGGKPGGEGSGKPGGKPGSGEGGKPGGKPGEGSGGKPGGKPEGGSGGKPGGSGKPGGKPEGGGSGKPGGKPGEGGKPGGEGSGKPGGSGKPG W576GGSGKPGKPGGSGSGKPGSGKPGGGSGKPGSGKPGGGSGKPGSGKPGGGSGKPGS 787 23.4GKPGGGGKPGSGSGKPGGGKPGGSGGKPGGGSGKPGKPGSGGSGKPGSGKPGGGSGGKPGKPGSGGSGGKPGKPGSGGGSGKPGKPGSGGSGGKPGKPGSGGSGGKPGKPGSGGSGKPGSGKPGGGSGKPGSGKPGSGGSGKPGKPGSGGSGKPGSGKPGSGSGKPGSGKPGGGSGKPGSGKPGSGGSGKPGKPGSGGGKPGSGSGKPGGGKPGSGSGKPGGGKPGGSGGKPGGSGGKPGKPGSGGGSGKPGKPGSGGGSGKPGKPGGSGSGKPGSGKPGGGSGKPGSGKPGSGGSGKPGKPGSGGSGGKPGKPGSGGGKPGSGSGKPGGGKPGSGSGKPGGGKPGSGSGKPGGGKPGSGSGKPGGSGKPGSGKPGGGSGGKPGKPGSGGSGKPGSGKPGSGGSGKPGKPGGSGSGKPGSGKPGGGSGKPGSGKPGGGSGKPGSGKPGGGSGKPGSGKPGGGGKPGSGSGKPGGSGGKPGKPGSGGSGGKPGKPGSGGSGKPGSGKPGGGSGGKPGKPGSGG Y576GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGE 788 15.7GSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEGEGSGEGSEGEGGSEGSEGEGGGEGSEGEGSGEGSEGEGGSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSEGSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEGSGEGSEGEGGSEGGEGEGSEGGSEGEGSEGGSEGEGGEGSGEGEGGGEGSEGEGSEGSGEGEGSGEGSE AD576GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGG 789 13.6PGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSEGSSGPGESS AE576AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 790  6.1APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AF540GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGP 791  8.8GTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAP AF504GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS 792  7.0PGSNPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP AE864GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA 793  6.1PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTREGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTREGTSTEPSEGSAP AF864GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASP 794  7.5GTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP AG868GGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSG 795  7.5ATGSPGSNPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP AM875GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSAS 796  4.5PGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP AM1318GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSAS 797  4.5PGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP

Example 59: Calculation of TEPITOPE Scores

TEPITOPE scores of 9mer peptide sequence can be calculated by addingpocket potentials as described by Sturniolo [Sturniolo, T., et al.(1999) Nat Biotechnol, 17: 555]. In the present Example, separateTepitope scores were calculated for individual HLA alleles. Table 32shows as an example the pocket potentials for HLA*0101B, which occurs inhigh frequency in the Caucasian population. To calculate the TEPITOPEscore of a peptide with sequence P1-P2-P3-P4-P5-P6-P7-P8-P9, thecorresponding individual pocket potentials in Table 32 were added. TheHLA*0101B score of a 9mer peptide with the sequence FDKLPRTSG (SEQ IDNO: 798) would be the sum of 0, −1.3, 0, 0.9, 0, −1.8, 0.09, 0, 0.

To evaluate the TEPITOPE scores for long peptides one can repeat theprocess for all 9mer subsequences of the sequences. This process can berepeated for the proteins encoded by other HLA alleles. Tables 33-36give pocket potentials for the protein products of HLA alleles thatoccur with high frequency in the Caucasian population.

TEPITOPE scores calculated by this method range from approximately −10to +10. However, 9mer peptides that lack a hydrophobic amino acid(FKLMVWY (SEQ ID NO: 799)) in P1 position have calculated TEPITOPEscores in the range of −1009 to −989. This value is biologicallymeaningless and reflects the fact that a hydrophobic amino acid servesas an anchor residue for HLA binding and peptides lacking a hydrophobicresidue in P1 are considered non binders to HLA. Because most XTENsequences lack hydrophobic residues, all combinations of 9mersubsequences will have TEPITOPEs in the range in the range of −1009 to−989. This method confirms that XTEN polypeptides may have few or nopredicted T-cell epitopes.

TABLE 32 Pocket potential for HLA*0101B allele. Amino Acid P1 P2 P3 P4P5 P6 P7 P8 P9 A −999 0 0 0 — 0 0 — 0 C −999 0 0 0 — 0 0 — 0 D −999 −1.3−1.3 −2.4 — −2.7 −2 — −1.9 E −999 0.1 −1.2 −0.4 — −2.4 −0.6 — −1.9 F 00.8 0.8 0.08 — −2.1 0.3 — −0.4 G −999 0.5 0.2 −0.7 — −0.3 −1.1 — −0.8 H−999 0.8 0.2 −0.7 — −2.2 0.1 — −1.1 I −1 1.1 1.5 0.5 — −1.9 0.6 — 0.7 K−999 1.1 0 −2.1 — −2 −0.2 — −1.7 L −1 1 1 0.9 — −2 0.3 — 0.5 M −1 1.11.4 0.8 — −1.8 0.09 — 0.08 N −999 0.8 0.5 0.04 — −1.1 0.1 — −1.2 P −999−0.5 0.3 −1.9 — −0.2 0.07 — −1.1 Q −999 1.2 0 0.1 — −1.8 0.2 — −1.6 R−999 2.2 0.7 −2.1 — −1.8 0.09 — −1 S −999 −0.3 0.2 −0.7 — −0.6 −0.2 —−0.3 T −999 0 0 −1 — −1.2 0.09 — −0.2 V −1 2.1 0.5 −0.1 — −1.1 0.7 — 0.3W 0 −0.1 0 −1.8 — −2.4 −0.1 — −1.4 Y 0 0.9 0.8 −1.1 — −2 0.5 — −0.9

TABLE 33 Pocket potential for HLA*0301B allele. Amino acid P1 P2 P3 P4P5 P6 P7 P8 P9 A −999 0 0 0 — 0 0 — 0 C −999 0 0 0 — 0 0 — 0 D −999 −1.3−1.3 2.3 — −2.4 −0.6 — −0.6 E −999 0.1 −1.2 −1 — −1.4 −0.2 — −0.3 F −10.8 0.8 −1 — −1.4 0.5 — 0.9 G −999 0.5 0.2 0.5 — −0.7 0.1 — 0.4 H −9990.8 0.2 0 — −0.1 −0.8 — −0.5 I 0 1.1 1.5 0.5 — 0.7 0.4 — 0.6 K −999 1.10 −1 — 1.3 −0.9 — −0.2 L 0 1 1 0 — 0.2 0.2 — −0 M 0 1.1 1.4 0 — −0.9 1.1— 1.1 N −999 0.8 0.5 0.2 — −0.6 −0.1 — −0.6 P −999 −0.5 0.3 −1 — 0.5 0.7— −0.3 Q −999 1.2 0 0 — −0.3 −0.1 — −0.2 R −999 2.2 0.7 −1 — 1 −0.9 —0.5 S −999 −0.3 0.2 0.7 — −0.1 0.07 — 1.1 T −999 0 0 −1 — 0.8 −0.1 —−0.5 V 0 2.1 0.5 0 — 1.2 0.2 — 0.3 W −1 −0.1 0 −1 — −1.4 −0.6 — −1 Y −10.9 0.8 −1 — −1.4 −0.1 — 0.3

TABLE 34 Pocket potential for HLA*0401B allele. Amino acid P1 P2 P3 P4P5 P6 P7 P8 P9 A −999 0 0 0 — 0 0 — 0 C −999 0 0 0 — 0 0 — 0 D −999 −1.3−1.3 1.4 — −1.1 −0.3 — −1.7 E −999 0.1 −1.2 1.5 — −2.4 0.2 — −1.7 F 00.8 0.8 −0.9 — −1.1 −1 — −1 G −999 0.5 0.2 −1.6 — −1.5 −1.3 — −1 H −9990.8 0.2 1.1 — −1.4 0 — 0.08 I −1 1.1 1.5 0.8 — −0.1 0.08 — −0.3 K −9991.1 0 −1.7 — −2.4 −0.3 — −0.3 L −1 1 1 0.8 — −1.1 0.7 — −1 M −1 1.1 1.40.9 — −1.1 0.8 — −0.4 N −999 0.8 0.5 0.9 — 1.3 0.6 — −1.4 P −999 −0.50.3 −1.6 — 0 −0.7 — −1.3 Q −999 1.2 0 0.8 — −1.5 0 — 0.5 R −999 2.2 0.7−1.9 — −2.4 −1.2 — −1 S −999 −0.3 0.2 0.8 — 1 −0.2 — 0.7 T −999 0 0 0.7— 1.9 −0.1 — −1.2 V −1 2.1 0.5 −0.9 — 0.9 0.08 — −0.7 W 0 −0.1 0 −1.2 —−1 −1.4 — −1 Y 0 0.9 0.8 −1.6 — −1.5 −1.2 — −1

TABLE 35 Pocket potential for HLA*0701B allele. Amino acid P1 P2 P3 P4P5 P6 P7 P8 P9 A −999 0 0 0 — 0 0 — 0 C −999 0 0 0 — 0 0 — 0 D −999 −1.3−1.3 −1.6 — −2.5 −1.3 — −1.2 E −999 0.1 −1.2 −1.4 — −2.5 0.9 — −0.3 F 00.8 0.8 0.2 — −0.8 2.1 — 2.1 G −999 0.5 0.2 −1.1 — −0.6 0 — −0.6 H −9990.8 0.2 0.1 — −0.8 0.9 — −0.2 I −1 1.1 1.5 1.1 — −0.5 2.4 — 3.4 K −9991.1 0 −1.3 — −1.1 0.5 — −1.1 L −1 1 1 −0.8 — −0.9 2.2 — 3.4 M −1 1.1 1.4−0.4 — −0.8 1.8 — 2 N −999 0.8 0.5 −1.1 — −0.6 1.4 — −0.5 P −999 −0.50.3 −1.2 — −0.5 −0.2 — −0.6 Q −999 1.2 0 −1.5 — −1.1 1.1 — −0.9 R −9992.2 0.7 −1.1 — −1.1 0.7 — −0.8 S −999 −0.3 0.2 1.5 — 0.6 0.4 — −0.3 T−999 0 0 1.4 — −0.1 0.9 — 0.4 V −1 2.1 0.5 0.9 — 0.1 1.6 — 2 W 0 −0.1 0−1.1 — −0.9 1.4 — 0.8 Y 0 0.9 0.8 −0.9 — −1 1.7 — 1.1

TABLE 36 Pocket potential for HLA*1501B allele. Amino acid P1 P2 P3 P4P5 P6 P7 P8 P9 A −999 0 0 0 — 0 0 — 0 C −999 0 0 0 — 0 0 — 0 D −999 −1.3−1.3 −0.4 — −0.4 −0.7 — −1.9 E −999 0.1 −1.2 −0.6 — −1 −0.7 — −1.9 F −10.8 0.8 2.4 — −0.3 1.4 — −0.4 G −999 0.5 0.2 0 — 0.5 0 — −0.8 H −999 0.80.2 1.1 — −0.5 0.6 — −1.1 I 0 1.1 1.5 0.6 — 0.05 1.5 — 0.7 K −999 1.1 0−0.7 — −0.3 −0.3 — −1.7 L 0 1 1 0.5 — 0.2 1.9 — 0.5 M 0 1.1 1.4 1 — 0.11.7 — 0.08 N −999 0.8 0.5 −0.2 — 0.7 0.7 — −1.2 P −999 −0.5 0.3 −0.3 —−0.2 0.3 — −1.1 Q −999 1.2 0 −0.8 — −0.8 −0.3 — −1.6 R −999 2.2 0.7 0.2— 1 −0.5 — −1 S −999 −0.3 0.2 −0.3 — 0.6 0.3 — −0.3 T −999 0 0 −0.3 — −00.2 — −0.2 V 0 2.1 0.5 0.2 — −0.3 0.3 — 0.3 W −1 −0.1 0 0.4 — −0.4 0.6 —−1.4 Y −1 0.9 0.8 2.5 — 0.4 0.7 — −0.9

Example 60: Analytical Size Exclusion Chromatography of XTEN FusionProteins with Diverse Payloads

Size exclusion chromatography analyses were performed on fusion proteinscontaining various therapeutic proteins and unstructured recombinantproteins of increasing length. An exemplary assay used a TSKGel-G4000SWXL (7.8 mm×30 cm) column in which 40 μg of purified glucagon fusionprotein at a concentration of 1 mg/ml was separated at a flow rate of0.6 ml/min in 20 mM phosphate pH 6.8, 114 mM NaCl. Chromatogram profileswere monitored using OD214 nm and OD280 nm. Column calibration for allassays were performed using a size exclusion calibration standard fromBioRad; the markers include thyroglobulin (670 kDa), bovinegamma-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobuin(17 kDa) and vitamin B12 (1.35 kDa). Representative chromatographicprofiles of Glucagon-Y288, Glucagon-Y144, Glucagon-Y72, Glucagon-Y36 areshown as an overlay in FIG. 32. The data show that the apparentmolecular weight of each compound is proportional to the length of theattached XTEN sequence. However, the data also show that the apparentmolecular weight of each construct is significantly larger than thatexpected for a globular protein (as shown by comparison to the standardproteins run in the same assay). Based on the SEC analyses for allconstructs evaluated, the apparent molecular weights, the apparentmolecular weight factor (expressed as the ratio of apparent molecularweight to the calculated molecular weight) and the hydrodynamic radius(R_(H) in nm) are shown in Table 37. The results indicate thatincorporation of different XTENs of 576 amino acids or greater confersan apparent molecular weight for the fusion protein of approximately 339kDa to 760, and that XTEN of 864 amino acids or greater confers anapparent molecular weight greater than approximately 800 kDA. Theresults of proportional increases in apparent molecular weight to actualmolecular weight were consistent for fusion proteins created with XTENfrom several different motif families; i.e., AD, AE, AF, AG, and AM,with increases of at least four-fold and ratios as high as about17-fold. Additionally, the incorporation of XTEN fusion partners with576 amino acids or more into fusion proteins with the various payloads(and 288 residues in the case of glucagon fused to Y288) resulted with ahydrodynamic radius of 7 nm or greater; well beyond the glomerular poresize of approximately 3-5 nm. Accordingly, it is expected that fusionproteins comprising growth and XTEN have reduced renal clearance,contributing to increased terminal half-life and improving thetherapeutic or biologic effect relative to a corresponding un-fusedbiologic payload protein.

TABLE 37 SEC analysis of various polypeptides XTEN Apparent Con- orThera- Actual Apparent Molecular struct fusion peutic MW MW Weight R_(H)Name partner Protein (kDa) (kDa) Factor (nm) AC14 Y288 Glucagon 28.7 37012.9 7.0 AC28 Y144 Glucagon 16.1 117 7.3 5.0 AC34 Y72 Glucagon 9.9 58.65.9 3.8 AC33 Y36 Glucagon 6.8 29.4 4.3 2.6 AC89 AF120 Glucagon 14.1 76.45.4 4.3 AC88 AF108 Glucagon 13.1 61.2 4.7 3.9 AC73 AF144 Glucagon 16.395.2 5.8 4.7 AC53 AG576 GFP 74.9 339 4.5 7.0 AC39 AD576 GFP 76.4 546 7.17.7 AC41 AE576 GFP 80.4 760 9.5 8.3 AC52 AF576 GFP 78.3 526 6.7 7.6AC398 AE288 FVII 76.3 650 8.5 8.2 AC404 AE864 FVII 129 1900 14.7 10.1AC85 AE864 Exendin-4 83.6 938 11.2 8.9 AC114 AM875 Exendin-4 82.4 134416.3 9.4 AC143 AM875 hGH 100.6 846 8.4 8.7 AC227 AM875 IL-1ra 95.4 110311.6 9.2 AC228 AM1318 IL-1ra 134.8 2286 17.0 10.5

Example 61: Construction of CBD-XTEN-Cys, a Cysteine-Engineered XTEN

A Cysteine Island (CysIsland) encoding the amino acid sequenceGGSPAGSCTSP (SEQ ID NO: 174) containing one cysteine was introduced byannealed oligos in the CBD-stuffer-GFP vector to obtainCBD-CysIsland-GFP, where CysIsland is flanked by the restriction sitesBsaI and BbsI. The CBD-stuffer-GFP vector is a pET30 derivative fromNovagen with TEV protease recognition site between CBD and the stuffer.Constructs were previously generated by replacing the stuffer inCBD-stuffer-GFP vector with genes encoding XTEN_AE288 and XTEN_AE576.The plasmid of CBD-XTEN_AE288-GFP was digested with BsaI/NcoI togenerate the small fragment as the insert. The plasmid ofCBD-CysIsland-GFP was digested with BbsI/NcoI to generate the largefragment as the vector. The insert and vector fragments were ligated andthe ligation mixture was electroporated into BL21-Gold (DE3) cells toobtain transformants of CBD-CysIsland-XTEN_AE288-GFP Similarly, theplasmid of CBD-CysIsland-XTEN_AE288-GFP was digested with BsaI/NcoI togenerate the small fragment as the insert. The plasmid ofCBD-XTEN_AE576-GFP was digested with BbsI/NcoI to generate the largefragment as the vector. The insert and vector fragments were ligated andthe ligation mixture was electroporated into BL21-Gold (DE3) cells toobtain transformants of CBD-XTEN_AE576-CysIsland-XTEN_AE288-GFP.Finally, the plasmid of CBD-XTEN_AE576-CysIsland-XTEN_AE288-GFP wasdigested with BbsI/HindIII to remove GFP and ligate with annealed oligosfor the stop codon, and the ligation mixture was electroporated intoBL21-Gold (DE3) cells to obtain transformants ofCBD-XTEN_AE576-CysIsland-XTEN_AE288, which has the DNA and encoded aminoacid sequences that follow in Table 38. Additional constructs can becreated with cysteines inserted at different locations within the XTENsequence by the selection of restriction sites appropriate for the givenlocation, including multiple insertions. The method could also beutilized to create lysine-engineered XTEN by substitution of codonsencoding lysine for those encoding cysteine in the oligonucleotides.

TABLE 38 DNA and amino acid sequence of Cys-engineered XTEN Clone SEQ IDAmino Acid SEQ ID Name DNA Sequence NO: Sequence NO: CBD-TEV-ATGGCAAATACACCGGTATCAGGCAATTTGAAGGTTGAA 800 MANTPVSGNLKV 801 AE576-TTCTACAACAGCAATCCTTCAGATACTACTAACTCAATCA EFYNSNPSDTTNS CysIsland-ATCCTCAGTTCAAGGTTACTAATACCGGAAGCAGTGCAA INPQFKVTNTGSS AE288TTGATTTGTCCAAACTCACATTGAGATATTATTATACAGT AIDLSKLTLRYYYAGACGGACAGAAAGATCAGACCTTCTGGGCTGACCATGC TVDGQKDQTFWTGCAATAATCGGCAGTAACGGCAGCTACAACGGAATTAC ADHAAIIGSNGSYTTCAAATGTAAAAGGAACATTTGTAAAAATGAGTTCCTC NGITSNVKGTFVAACAAATAACGCAGACACCTACCTTGAAATCAGCTTTAC KMSSSTNNADTYAGGCGGAACTCTTGAACCGGGTGCACATGTTCAGATACA LEISFTGGTLEPGAGGTAGATTTGCAAAGAATGACTGGAGTAACTATACACA AHVQIQGRFAKNGTCAAATGACTACTCATTCAAGTCTGCTTCACAGTTTGTT DWSNYTQSNDYSGAATGGGATCAGGTAACAGCATACTTGAACGGTGTTCTT FKSASQFVEWDQGTATGGGGTAAAGAACCCGGTGGCAGTGTAGTAGGTTCA VTAYLNGVLVWGGTTCAGGATCCGAAAATCTGTATTTTCAGGGTGGGTCTC GKEPGGSVVGSGCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAG SGSENLYFQGGSPGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTAC GSPAGSPTSTEEGCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCC TSESATPESGPGTAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACT STEPSEGSAPGSPGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAA AGSPTSTEEGTSTCCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTA EPSEGSAPGTSTECCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTG PSEGSAPGTSESAGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTC TPESGPGSEPATSTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACT GSETPGSEPATSGGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGC SETPGSPAGSPTSCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA TEEGTSESATPESGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT GPGTSTEPSEGSAAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTT PGTSTEPSEGSAPCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTA GSPAGSPTSTEEGCTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAA TSTEPSEGSAPGTGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACC STEPSEGSAPGTSGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAAC ESATPESGPGTSTCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGC EPSEGSAPGTSESTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTA ATPESGPGSEPATGCGCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGCG SGSETPGTSTEPSCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCC EGSAPGTSTEPSECAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAG GSAPGTSESATPEGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTA SGPGTSESATPESCCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCG GPGSPAGSPTSTEAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTG EGTSESATPESGPAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTG GSEPATSGSETPGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACC TSESATPESGPGTGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTC STEPSEGSAPGTSCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGA TEPSEGSAPGTSTGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGG EPSEGSAPGTSTETAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAG PSEGSAPGTSTEPCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGA SEGSAPGTSTEPSGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACC EGSAPGSPAGSPTAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGG STREGTSTEPSEGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACC SAPGTSESATPESTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAA GPGSEPATSGSETCCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAA PGTSESATPESGPGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACC GSEPATSGSETPGGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTAC TSESATPESGPGTTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACT STEPSEGSAPGTSTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTA ESATPESGPGSPACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACTG GSPTSTEEGSPAGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCC SPTSTEEGSPAGSCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAG PTSTEEGTSESATGTGGTAGCCCGGCTGGCTCTTGTACCTCTCCAGGTACCTC PESGPGTSTEPSETGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACC GSAPGGSPAGSCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGC TSPGTSESATPESGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACC GPGSEPATSGSETTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTC PGTSESATPESGPCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGG GSEPATSGSETPGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACC TSESATPESGPGTGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGC STEPSEGSAPGSPCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCA AGSPTSTEEGTSEGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTA SATPESGPGSEPAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCC TSGSETPGTSESACGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTAC TPESGPGSPAGSPCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAG TSTEEGSPAGSPTCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCT STEEGTSTEPSEGACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCC SAPGTSESATPESCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTC GPGTSESATPESGTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGA PGTSESATPESGPAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGA GSEPATSGSETPGGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACC SEPATSGSETPGSAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGT PAGSPTSTEEGTSAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC TEPSEGSAPGTSTTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA EPSEGSAPGSEPACTGAACCGTCCGAGGGCAGCGCACCAGGTTAA TSGSETPGTSESA TPESGPGTSTEPS EGSAPG

Example 62: Purification of CBD-XTEN-Cys

E. coli containing AC292 on a plasmid were grown to saturation overnightin 2×YT and then 200 ml of this culture was used to inoculate a 25 Lculture of 2×YT media in a wavebag. Both cultures were in the presenceof 50 μg/ml kanamycin. The second culture was grown to an OD600 of ˜1.0at 37° C., chilled to 26° C., and induced with 12 ml of 1M IPTGovernight. The cell pellet was harvested at 4000 rpm in a SLA-3000 rotorspinning for 20 minutes. The cell pellet (184 g) was resuspended in 736ml of 20 mM Tris pH 6.8, 50 mM NaCl. The resuspended cells were lysedwith a microfluidizer at 20,000 psi and then heated to 75° C. for 15minutes, followed by rapid cooling on ice for 30 minutes. The lysate wasthen clarified by centrifugation. The clarified lysate was then loadedon to a DE52 column, previously sanitized with NaOH and equilibratedwith 20 mM Tris pH 6.8, 50 mM NaCl. The column was washed with 5 columnvolumes of 20 mM Tris pH 6.8, 50 mM NaCl, 5 column volumes of 20 mM TrispH 6.8, 150 mM NaCl and eluted with 5 column volumes of 20 mM Tris pH6.8, 250 mM NaCl. The pooled elution fractions, were then loaded on to amacrocapQ column, previously sanitized with NaOH and equilibrated with20 mM Tris pH 6.8, 50 mM NaCl. The column was washed with 9 columnvolumes of 20 mM Tris pH 6.8, 50 mM NaCl, 9 column volumes of 20 mM TrispH 6.8, 100 mM NaCl and eluted with 9 column volumes of 20 mM Tris pH6.8, 250 mM NaCl. The pooled elution fractions were adjusted to a 15%w/v sodium sulfate and then loaded on to a octyl sepharose FF column,previously sanitized with NaOH and equilibrated with Tris pH 7.5. Thecolumn was washed with 4 column volumes of 20 mM Tris pH 7.5 15% w/vsodium sulfate, and eluted with 4 column volumes of 20 mM Tris pH 7.5,5% w/v sodium sulfate. The sample was stored at 4° C. and given the lot#AP197. The purified cysteine-engineered XTEN could then serve as asuitable reactant for conjugation with a drug, such as a drug from Table5, resulting in an XTEN-drug conjugate.

Example 63: Conjugation and Purification of FITC-X-XTEN

Purified protein derived from AC272, lot #AP197, was labeled with FITCmaleimide. The sample was reduced by incubating at room temperature with5 mM TCEP for 1 hour. The sample was then desalted into PBS using DG-10columns. The sample was labeled by adding a 25-fold molar excess ofFITC-maleimide in DMSO and incubating at room temperature for 2 hours.Note that the volume adjusted such that the DMSO concentration was <5%of total solvent. The reaction was quenched by adding 2 mM DTT and thenthe sample was digested overnight with TEV protease. The sample wasdiluted two fold with 20 mM Tris pH 7.5 and loaded onto a macrocapQcolumn, previously sanitized with NaOH and equilibrated with 20 mM TrispH 7.5. The column was washed with 5 column volumes of 20 mM Tris pH7.5, 135 mM NaCl, 5 column volumes of 20 mM Tris pH 7.5, 175 mM NaCl andeluted with 5 column volumes of 20 mM Tris pH 7.5, 250 mM NaCl. Thepooled elution fractions were then digested with TEV over 60 hours at 4C to complete the digestion. The digested samples were then twice passedover a 1 ml perloza column previously sanitized with NaOH andequilibrated with 20 mM Tris pH 7.5, 135 mM NaCl. To remove any freeFITC the sample was then dialyzed against 20 mM Tris pH 7.5, 135 mM NaClusing a 10,000 MWCO membrane. Co-migration of the OD214 protein signaland OD495 FITC signal in a SEC column indicate successful conjugation ofthe XTEN with the label, with minimal free dye contamination (FIG. 42B).The successful conjugation is also indicated by apparent large MW of theprotein with FITC fluorescence in SDS PAGE (FIG. 42A).

Example 64: Purification of GFP-X-XTEN

GFP (AC219) was chemically cross-linked to XTEN by a bifunctionalcross-linker with an amine reactive group to couple to the GFP lysinesand a cysteine reactive group to couple to the free cysteine engineeredinto the XTEN in AC292. GFP was labeled with bi-functional cross linkersulfo-SMCC by incubating at room temperature for 2 hours. The proteinwas desalted into PBS using DG-10 columns to remove free sulfo-SMCC.Purified protein derived from AC272, lot #AP197 was reduced and desaltedinto PBS on DG-10 columns and mixed with the labeled GFP to allow forcrosslinking. The crosslinking reaction was quenched with 2 mM DTT andTEV added to remove the CBD domain in a overnight incubation at 4° C.The following day additional TEV was added to complete the digestionwith an additional 60 hour 4° C. incubation. Following TEV digestion thesample was dilute to 100 ml in 20 mM Tris pH 7.5 and loaded onto amacrocapQ column, previously sanitized with NaOH and equilibrated with20 mM Tris pH 7.5. The column was washed with 5 column volumes of 20 mMTris pH 7.5, 5 column volumes of 20 mM Tris pH 7.5, 50 mM NaCl, 5 columnvolumes of 20 mM Tris pH 7.5, 100 mM NaCl, 5 column volumes of 20 mMTris pH 7.5, 150 mM NaCl, 5 column volumes of 20 mM Tris pH 7.5, 200 mMNaCl, 5 column volumes of 20 mM Tris pH 7.5, 250 mM NaCl, 5 columnvolumes of 20 mM Tris pH 7.5, 300 mM NaCl, and 5 column volumes of 20 mMTris pH 7.5, 500 mM NaCl. The peak elution fractions were pooled andstored at 4° C. Crosslinking was confirm by co-migration of the OD214protein signal and OD395 GFP signal in a SEC column, with the SEC outputshown as overlays in FIG. 43.

Example 65: Pharmacokinetics of GFP-XTEN and FITC-XTEN Conjugates

The pharmacokinetics of the GFP-XTEN and FITC-XTEN cross-linkedconjugates prepared as described in the Examples above were tested incynomolgus monkeys. GFP-XTEN and FITC-XTEN were administered to malecynos IV at 2 mg/kg and dose volumes of 0.77 and 0.68 mL respectively.Blood samples (1.0 mL) were collected into prechilled heparinized tubesat predose, 2, 4, 8, 24, 48, 72, 96, 120, 168, 216, 264, 336, 388, 432,504 hour time points, and processed into plasma. Quantitation wasperformed by ELISA assay using the anti-XTEN antibody for both captureand detection in the case of GFP-XTEN and anti-XTEN capture andanti-FITC detection in the case of FITC-XTEN. A non-compartmentalanalysis was performed in WinNonLin with all time points included in thefit to determine the PK parameters. The pharmacokinetic results aresummarized in Table 39 and FIG. 44. The data show XTEN can extend thehalf-life of molecules to which it is chemically conjugated in a mannercomparable to genetic fusions to payloads of similar size.

TABLE 39 PK parameters: Construct Cmax (ng/mL) AUC (hr*ng/mL) T ½ (hrs)GFP-X-XTEN (AP197d) 52800 8220000 107 FITC-X-XTEN AP197e 18900 393000084.2

Example 66: Preparation of Anti-Her2-XTEN-Paclitaxel BFP-D Conjugate

The conjugation process that can be employed is generically illustratedin FIG. 45. Drugs can be conjugated to XTEN using the methods of thedisclosure or those, e.g., of US Patent publication No. 2009/0074704. Bythe methods, paclitaxel can first be reacted with an activated linker toa suitable functional group, as shown in FIG. 45. The resultingconjugate can be purified by suitable, standard methods known in theart, including HPLC, size exclusion chromatography, gel filtration, ionexchange chromatography, or combinations thereof. Subsequently, theactivated paclitaxel conjugate would be incubated with aHer2-XTEN-Cysthat was expressed and purified as described in Example 24, using atleast a 25-fold excess of paclitaxel conjugate to ensure all reactivecysteine sites on the XTEN are conjugated. The conjugation could beperformed essentially as described in Example 64. Excess paclicaxelwould be removed from the reaction mixture by dialysis and the finalpurified product would be concentrated and stored for subsequent use.

Example 67: Clinical Applications of Anti-Her2-XTEN-PactlitaxelCompositions

Her2 antigen is overexpressed on a large number of solid malignancies.Expression is particularly high on many breast cancer cells. Herceptinhas been approved for the treatment of Her2-positive breast cancers. Theinvention contemplates that anti-Her2-XTEN-paclitaxel can be used forthe treatment of the same patient population. Clinical trials can bedesigned such that the efficacy and advantages of theanti-Her2-XTEN-paclitaxel compositions can be verified in humans. Suchstudies in patients would comprise three phases. First, a Phase I safetyand pharmacokinetics study in adult patients would be conducted todetermine the maximum tolerated dose and pharmacokinetics andpharmacodynamics in humans. These studies define potential toxicitiesand adverse events to be tracked in future studies. The scheme of thestudy would be to use single escalating doses of aHer2-XTEN-paclitaxelcompositions and measure the biochemical, PK, and clinical parameters.This would permit the determination of the maximum tolerated dose andestablish the threshold and maximum concentrations in dosage andcirculating drug that constitute the therapeutic window to be used insubsequent Phase II and Phase III trials conducted in target indicationsto determine efficacy and tolerability of the aHER2-XTEN-paclitaxelcompositions.

TABLE 40Binding fusion proteins with targeting moieties to single targets SEQ IDName* Protein Sequence NO: CTLA4_4-MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMM 802 AM875GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP AE912-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTST 803 CTLA4EEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMNIGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEG CTLA4-MAMHVAQPAVVLASSRGIASFVCEYASPGKAILVRVTVLRQADSQVTEVCAATY 804 AE36-MMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGN CTLA4-GTQIYVIDPEGAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGPAMHVAQP AE864AVVLASSRGIASFVCEYASPGKAILVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG CTLA4-MAMHVAQPAVVLASSRGIASFVCEYASPGKAIEVRVTVLRQADSQVTEVCAATY 805 AE158-MMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGN CTLA4-GTQIYVIDPEGAPSTGGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP AE864ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVIEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG CTLA4-MAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATY 806 AE158-MMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGN CTLA4-GTQIYVIDPEPCPDSGAPSTGGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTS AE864ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAMHVAQPAVVLASSRGIASFVCEYASPGKAIEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEP CPDSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AE912-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTST 807 aIL6REEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEhPSEGSAPGADIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKTGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGSQVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSS aIL6R-MADIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLH 808 AE864SGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKTGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGSQVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS APG AE912-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTST 809 aIL6R-EEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS AE144APGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGADIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKTGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGSQVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSGGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS AE48-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPGDIQMT 810 aIL6R-QSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFS AE864GSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKTGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGSQVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG aIL6R_VL-DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSG 811 AF144-VPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKGGTSTPESG aIL6R_VH-SASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAE AM875SPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGQVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPS EGSAPGAM923- MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSEGS 812aIL6R_VH- APGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAF144- APGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESaIL6R_VL GPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGQVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSGGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKG aIL6R_VL-DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSG 813 Linker_VPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKGGAPGTSE AE42-SATPESGPGSEPATSGSETPGTSTEPSEGSAPGPAGQVQLQESGPGLVRPSQTLSLTC aIL6R_VH-TVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQF AD576SLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSGGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSGSESGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSEGSSGPGESSG AM923-MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSEGS 814aIL6R_VH- APGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTLinker_ APGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESAM150- GPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSaIL6R_VH APGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGQVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSGGAPSTGGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAGQVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSG aIL6R_VL-DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSG 815 AE42-VPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKGGAPGTSE aIL6R_VH-SATPESGPGSEPATSGSETPGTSTEPSEGSAPGPAGQVQLQESGPGLVRPSQTLSLTC AM875TVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG AM923-MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSEGS 816aIL6R_VH- APGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAE42- APGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESaCD40_VL GPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGQVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSGGAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGPAGMAEIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIYYASHSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHGHSYPW TFGGGTKVEIKGaIL6R_VL- DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSG 817Y32- VPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKGTGSGEGSaIL6R_VH- EGEGGGEGSEGEGSGEGGEGEGSGSGQVQLQESGPGLVRPSQTLSLTCTVSGYSITSAM1296 DHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG aCD40-MAEIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIYYASHSIS 818 AE864GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHGHSYPWTFGGGTKVEIKTGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGTQVQLVQSGSELKKPGASVKVSCKASGYAFTTTGMQWVRQAPGQGLEWMGWINTHSGVPKYVEDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARSGNGNYDLAYFKYWGQGTLVTVSSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPAE912- MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTST 819aCD40 EEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIYYASHSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHGHSYPWTFGGGTKVEIKTGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGTQVQLVQSGSELKKPGASVKVSCKASGYAFTTTGMQWVRQAPGQGLEWMGWINTHSGVPKYVEDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARSGNGNYDLAYFKYWGQGTLVTVS AE912-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTST 820aCD40_VH- EEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAF144- APGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESaCD40_VL GPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQVQLVQSGSELKKPGASVKVSCKASGYAFTTTGMQWVRQAPGQGLEWMGWINTHSGVPKYVEDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARSGNGNYDLAYFKYWGQGTLVTVSSGGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGMAEIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIYYASHSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHGHSYPWTFGGGTKVEIKG aCD40_VL-MAEIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIYYASHSIS 821 AE144-GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHGHSYPWTFGGGTKVEIKGGSEPAT aCD40_VH-SGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT AE576SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGQVQLVQSGSELKKPGASVKVSCKASGYAFTTTGMQWVRQAPGQGLEWMGWINTHSGVPKYVEDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARSGNGNYDLAYFKYWGQGTLVTVSSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGT STEPSEGSAPGaCD40_VL- MAEIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIYYASHSIS 822AE42- GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHGHSYPWTFGGGTKVEIKGGAPGTSaCD40_VH- ESATPESGPGSEPATSGSETPGTSTEPSEGSAPGPAGQVQLVQSGSELKKPGASVKVSBC864 CKASGYAFTTTGMQWVRQAPGQGLEWMGWINTHSGVPKYVEDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARSGNGNYDLAYFKYWGQGTLVTVSSGGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSGASEPTSTEPGTSEPSTSEPGAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSEPSTSEPGAGSGASEPTSTEPGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAG AM923-MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSEGS 823aCD40_VH- APGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAE42- APGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPES BD864GPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGQVQLVQSGSELKKPGASVKVSCKASGYAFTTTGMQWVRQAPGQGLEWMGWINTHSGVPKYVEDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARSGNGNYDLAYFKYWGQGTLVTVSSGGAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGPAGGSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGSTAGSETSTEAGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAG aCD40_VL-MAEIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIYYASHSIS 824 Y32-GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHGHSYPWTFGGGTKVEIKGTGSGEG aCD40_VH-SEGEGGGEGSEGEGSGEGGEGEGSGSGQVQLVQSGSELKKPGASVKVSCKASGYAF AE576TTTGMQWVRQAPGQGLEWMGWINTHSGVPKYVEDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARSGNGNYDLAYFKYWGQGTLVTVSSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG AE912-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTST 825aCD40_VH- EEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAF144- APGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESaCD40_VL GPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQVQLVQSGSELKKPGASVKVSCKASGYAFTTTGMQWVRQAPGQGLEWMGWINTHSGVPKYVEDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARSGNGNYDLAYFKYWGQGTLVTVSSGGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGMAEIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIYYASHSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHGHSYPWTFGGGTKVEIKG aCD40_VL-MAEIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIYYASHSIS 826 AE144-GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHGHSYPWTFGGGTKVEIKGGSEPAT aCD40_VH-SGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT AE576SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGQVQLVQSGSELKKPGASVKVSCKASGYAFTTTGMQWVRQAPGQGLEWMGWINTHSGVPKYVEDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARSGNGNYDLAYFKYWGQGTLVTVSSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGT STEPSEGSAPGaHER2- MEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFL 827AE864 YSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKTGSGEGSGEGGGEGSEGEGSGEGGEGEGSGTEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST EPSEGSAPAE912- MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTST 828aHER2 EEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSSSLDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKTGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGTEVQLVESGGGLVQPGGSLRLSCASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS aHer2_VL-MEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFL 829 AE42-YSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGAPG aHer2_VH-TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGPAGEVQLVESGGGLVQPGGSLR AM1296LSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG AE912-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTST 830aHER2_VL- EEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAF144- APGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESaHER2_VH GPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGMEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSG AE48-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGEVQLVESG 831 aHER2_VH-GGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS AE144-VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVT aHER2_VL-VSGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEG AE576SAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGMEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA PGaHer2_VH_ EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG 8321-AE288- YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWaHer2_VL_ GQGTLVTVSGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG1-AF576 TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGMEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPG aEGFR-MEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGI 833 Y576PSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKTGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGTQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSGGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEGEGSGEGSEGEGGSEGSEGEGGGEGSEGEGSGEGSEGEGGSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSEGSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEGSGEGSEGEGGSEGGEGEGSEGGSEGEGSEGGSEGEGGEGSGEGEGGGEGSEGEGSEGSGEGEGSGEGSEG aEGFR_VL-MEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGI 834 AE42-PSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGAPGTSE aEGFR_VH-SATPESGPGSEPATSGSETPGTSTEPSEGSAPGPAGQVQLKQSGPGLVQPSQSLSITCT AD836VSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSGGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSSG AM923-MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSEGS 835aEGFR_VL- APGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAM150- APGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESaEGFR_VH GPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS 1APGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEhEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGMEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGAPSTGGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAGQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQG TLVTVSGaEGFR_VH- QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGN 836AF144- TDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGaEGFR_VL- TLVTVSGGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSEAF864 SPSGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGMEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP G AE912-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTST 837aEGFR_VH- EEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSY32- APGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESaEGFR_VL GPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSGTGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGSGMEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKG aEGFR_VL-MEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGI 838 Linker_PSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGTGSGEGS Y32-EGEGGGEGSEGEGSGEGGEGEGSGSGQVQLKQSGPGLVQPSQSLSITCTVSGFSLTN aEGFR_VH-YGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQ BC864SNDTAIYYCARALTYYDYEFAYWGQGTLVTVSGGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGIEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGIEPSGSEPATSGIEPSGTSEPSTSEPGAGSGASEPTSTEPGTSEPSTSEPGAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSEPATSGIEPSGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSEPSTSEPGAGSGASEPTSTEPGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGIEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSA G aEGFR_VH-QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGN 839 AE288-TDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQG aEGFR_VL-TLVTVSGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE BD864SATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGMEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGTPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGSTAGSETSTEAGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAG aCD3-Y288MKDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLA 840SGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGTQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYSARYYDDHYCLDYWGQGTPVTVSSGGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGSGEGSEGEGGSEGSEGEGSGEGSEGEGSEGGSEGEGGSEGSEGEGSGEGSEGEGGEGGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGS aCD3_VL-MKDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLA 841 AF144-SGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRGGTST aCD3_VH-PESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTS AE576STAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYSARYYDDHYCLDYWGQGTPVTVSSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS TEPSEGSAPGAM923- MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSEGS 842aCD3_VH- APGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAM150- APGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESaCD3_VL GPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYSARYYDDHYCLDYWGQGTPVTVSSGGAPSTGGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAGMKDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFT FGQGTKLQITRGaCD3_VL- MKDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLA 843Linker_ SGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRGGAPG AE4-2TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGPAGQVQLVQSGGGVVQPGRSLR aCD3_VH-LSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISTDKS AM875KSTAFLQMDSLRPEDTAVYYSARYYDDHYCLDYWGQGTPVTVSSGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG AE912-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTST 844 aCD3_VL-EEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS AE144-APGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES aCD3_VHGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGMKDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYSARYYDDHYCLDYWGQGTPVTVSSG AE48-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGQVQLVQSG 845 aCD3_VH-GGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQK Linker_FKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYSARYYDDHYCLDYWGQGTPVTVSS Y32-GTGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGSGMKDIQMTQSPSSLSASVGDRVT aCD3_VL-ITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSL AE864QPEDIATYYCQQWSSNPFTFGQGTKLQITRGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AM48-MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGMKDIQMTQ 846 aCD3_VL-SPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSG Linker_SGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRGGAPGTSESATPES AE42-GPGSEPATSGSETPGTSTEPSEGSAPGPAGQVQLVQSGGGVVQPGRSLRLSCKASGY aCD3_VH-TFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISTDKSKSTAFLQM AM875DSLRPEDTAVYYSARYYDDHYCLDYWGQGTPVTVSSGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG aEGRF_VHH-EVQLQESGGGLVQAGDSLRLSCLVSGRSFNSYTMGWFRQAPGKEREFVAAILWSGP 847 Linker_TTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAALGVLVLAPGNVY Y32-SYWGQGTQVTVSSAHHGTGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGSGEVQLQ aEGRF_VHHESGGGLVQAGDSLRLSCLVSGRSFNSYTMGWFRQAPGKEREFVAAILWSGPTTYYA 1-BC864DSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAALGVLVLAPGNVYSYWGQGTQVTVSSAHHGGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSGASEPTSTEPGTSEPSTSEPGAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSEPSTSEPGAGSGASEPTSTEPGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAG aEGRF_VHH-EVQLQESGGGLVQAGDSLRLSCLVSGRSFNSYTMGWFRQAPGKEREFVAAILWSGP 848 AE144-TTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAALGVLVLAPGNVY aEGRF_VHH-SYWGQGTQVTVSSAHHGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPA BD864GSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGEVQLQESGGGLVQAGDSLRLSCLVSGRSFNSYTMGWFRQAPGKEREFVAAILWSGPTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAALGVLVLAPGNVYSYWGQGTQVTVSSAHHGGSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGSTAGSETSTEAGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAG aEGRF_VHH-EVQLQESGGGLVQAGDSLRLSCLVSGRSFNSYTMGWFRQAPGKEREFVAAILWSGP 849 AF144-TTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAALGVLVLAPGNVY aEGRF_VHH-SYWGQGTQVTVSSAHHGGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSS AE576TAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGEVQLQESGGGLVQAGDSLRLSCLVSGRSFNSYTMGWFRQAPGKEREFVAAILWSGPTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAALGVLVLAPGNVYSYWGQGTQVTVSSAHHGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG aEGRF_VHH-EVQLQESGGGLVQAGDSLRLSCLVSGRSFNSYTMGWFRQAPGKEREFVAAILWSGP 850 Linker_TTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAALGVLVLAPGNVY AE42-SYWGQGTQVTVSSAHHGGAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPG aEGRF_VHH-PAGEVQLQESGGGLVQAGDSLRLSCLVSGRSFNSYTMGWFRQAPGKEREFVAAIL AM875WSGPTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAALGVLVLAPGNVYSYWGQGTQVTVSSAHHGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GaEGRF_VHH- EVQLQESGGGLVQAGDSLRLSCLVSGRSFNSYTMGWFRQAPGKEREFVAAILWSGP 851Linker_ TTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAALGVLVLAPGNVY AM150-SYWGQGTQVTVSSAHHGGAPSTGGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS aEGRF_VHH-APGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGS AM1296APGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAGEVQLQESGGGLVQAGDSLRLSCLVSGRSFNSYTMGWFRQAPGKEREFVAAILWSGPTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAALGVLVLAPGNVYSYWGQGTQVTVSSAHHGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG *″a″ before target protein name = anti

TABLE 41Binding fusion proteins with targeting moieties to different targetsSEQ ID Name* Protein Sequence NO: aHer2-MEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKWYSASFLYSGVPSRFSG 852Y288-SRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKTGSGEGSEGEGGGEGSEGEGSGEGaEGFR GEGEGSGTEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSGGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGSGEGSEGEGGSEGSEGEGSGEGSEGEGSEGGSEGEGGSEGSEGEGSGEGSEGEGGEGGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKTGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGTQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVS aHer2-MEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKWYSASFLYSGVPSRFSG 853Y288-SRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKTGSGEGSEGEGGGEGSEGEGSGEG aCD3GEGEGSGTEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSGSPGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGSGEGSEGEGGSEGSEGEGSGEGSEGEGSEGGSEGEGGSEGSEGEGSGEGSEGEGGEGGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGSSSLEGTKDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGTQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYSARYYDDHYCLDYWGQGTPVTVSSTSG aCD3_VLMKDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGV 854 1-AE48-PSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRGMAEPAGSPTS aCD3_VH-TEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGQVQLVQSGGGVVQPGRSLRLS AE144-CKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISTDKSKSTAF aHer2_VL-LQMDSLRPEDTAVYYSARYYDDHYCLDYWGQGTPVTVSSGGSEPATSGSETPGTSESA AE48-TPESGPGSEPATSGSETPGSPAGSPTSTEEGTSIEPSEGSAPGSEPATSGSETPGSEPATSGSaHer2_VH-ETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP AE864GMEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGMAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG aCD3_VH-QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYT 855 AE48-NYNQKFKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYSARYYDDHYCLDYWGQGTPVT aCD3_VL-VSSGMAEPAGSPTSSTEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGMKDIQMT AE144-QSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSG aHer2_VH-SGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRGGSEPATSGSETPGTSESA AE48-TPESGPGSEPATSGSETPGSPAGSPTSTEEGTSSTPSEGSAPGSEPATSGSETPGSEPATSGSaHer2_VL-ETPGSEPATSGSETPGTSSTPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP AE864GEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSGMAEPAGSPTSSTEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGMEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGSPAGSPTSTEEGTSESATPESGPGTSSTPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSSTEGTSSTPSEGSAPGTSSTPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSSTPSEGSAPGTSSTPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSSTPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSSTEGTSSTPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSSTPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSSTEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSSTEGTSSTPSEGSAPGTSSTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSSTPSEGSAPG aHer2_VL-MEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKWYSASFLYSG 856 AM48-VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGMAEPAGSPTS aHer2_VH-TEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGEVQLVESGGGLVQPGGSLRLS AF144-CAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTA aCD3_VL-YLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSGGTSTPESGSASPGTSP AM48-SGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGE aCD3_VH-SSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESST AM1296APGMKDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRGMAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYSARYYDDHYCLDYWGQGTPVTVSSGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSSTPSEGSAPGSPAGSPTSSTEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSSTLEGTSSTPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSSTPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSSTPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPGTSSTPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG aHer2_VH-EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT 857 AM48-RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT aHer2_VL-LVTVSGMAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGMEDIQ AF144-MTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF aCD3_VH-SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGTSTPESGSASPGTSP AM48-SGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGE aCD3_VL-SSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESST AM875APGQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYSARYYDDHYCLDYWGQGTPVTVSSGMAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGMKDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRGGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG aHer2_VL-MEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKWYSASFLYSG 858 Linker_VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGAPGTSESAT AE42-PESGPGSEPATSGSETPGTSTEPSEGSAPGPAGEVQLVESGGGLVQPGGSLRLSCAASGFN aHer2_VH-IKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSL AE288-RAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSGGTSESATPESGPGSEPATSGSETP aEGFR_GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS VL-ESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEP Linker_SEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGS AM150-ETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGP aEGFR_GTSTEPSEGSAPGMEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLL VH-AD576IKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGAPSTGGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAGQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSGGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSEGSSGPGESSG aHer2_VH-EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT 859 Linker_RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT AM150-LVTVSGGAPSTGGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT aHer2_VL-STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE AE288-PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAGMEDIQMTQSPSSLSASVGDRV aEGFR_TITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQP VH-EDFATYYCQQHYTTPPTFGQGTKVEIKGGTSESATPESGPGSEPATSGSETPGTSESATPE Linker_SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP AE42-GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS aEGFR_ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS VL-AE576PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSGGAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGPAGMEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG AE624-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEEG 860aHer2_VL-TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE Linker_SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPS AE42-EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESaHer2_VH-GPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPG AE288-TSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE aEGFR_SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS VL-EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE Linker_TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG AM150-SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGME aEGFR_DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVP VHSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGPAGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGMEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNINWPTTFGAGTKLELKGGAPSTGGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAGQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSG AE912-MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEEG 861aHer2_VH-TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE Linker_SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPS AM150-EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESaHer2_VL-GPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPG AE288-TSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE aEGFR_SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS VH-EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE Linker_TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG AE42-SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSE aEGFR_SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS VLEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSGGAPSTGGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAGMEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSGGAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGPAGMEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGT KLELKGAM923- MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSEGSAPG862 aEGFR_SEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTS VL-ESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSP Linker_TSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST AM150-EEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPG aEGFR_TSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTVH-AF144- PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATaHer2_VH- SGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPELinker_ SGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPAM150- GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSaHer2_VL EPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGMEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGAPSTGGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAGQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSGGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSGGAPSTGGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAGMEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKWYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKG *″a″ beforetarget moiety protein name = anti

What is claimed is:
 1. A binding fusion protein comprising: a firsttargeting moiety comprising a single-chain variable fragment (scFv) withspecific binding affinity to CD3; and a first extended recombinantpolypeptide (XTEN), wherein the first XTEN comprises a motif selectedfrom the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,and SEQ ID NO:
 9. 2. The binding fusion protein of claim 1, wherein thefirst XTEN comprises an amino acid sequence which has at least 90%sequence identity to a sequence selected from the group consisting ofAE144 (SEQ ID NO: 41), AE288 (SEQ ID NO: 47), AE576 (SEQ ID NO: 54) andAE864 (SEQ ID NO: 59).
 3. The binding fusion protein of claim 2, whereinthe first XTEN comprises an amino acid sequence selected from the groupconsisting of AE144 (SEQ ID NO: 41), AE288 (SEQ ID NO: 47), AE576 (SEQID NO: 54) and AE864 (SEQ ID NO: 59).
 4. The binding fusion protein ofclaim 1, wherein the first targeting moiety has specific bindingaffinity for an epsilon subunit of CD3 (CDR).
 5. The binding fusionprotein of claim 1, wherein the first XTEN is positioned N-terminal ofthe first targeting moiety.
 6. The binding fusion protein of claim 1,wherein the first XTEN is positioned C-terminal of the first targetingmoiety.
 7. The binding fusion protein of claim 1, further comprising asecond targeting moiety that differs from the first targeting moiety. 8.The binding fusion protein of claim 1, wherein the binding fusionprotein is configured according to formula I:(XTEN)_(x)-TM-(XTEN)_(y)   I wherein x is either 0 or 1; y is either 0or 1, wherein x+y≥1, and wherein TM is the first targeting moiety. 9.The binding fusion protein of claim 8, wherein x is 1 and y is
 1. 10.The binding fusion protein of claim 7, further comprising a second XTEN,wherein the second XTEN comprises a motif selected from the groupconsisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:9.
 11. The binding fusion protein of claim 10, wherein the second XTENcomprises an amino acid sequence which has at least 90% sequenceidentity to a sequence selected from the group consisting of AE144 (SEQID NO: 41), AE288 (SEQ ID NO: 47), AE576 (SEQ ID NO: 54) and AE864 (SEQID NO: 59).
 12. The binding fusion protein of claim 11, wherein thesecond XTEN comprises an amino acid sequence selected from the groupconsisting of AE144 (SEQ ID NO: 41), AE288 (SEQ ID NO: 47), AE576 (SEQID NO: 54) and AE864 (SEQ ID NO: 59).
 13. The binding fusion protein ofclaim 10, wherein the first XTEN is positioned at the N-terminus of thebinding fusion protein and the second XTEN is positioned at theC-terminus of binding fusion protein.
 14. A pharmaceutical compositioncomprising the binding fusion protein of claim 1, and at least onepharmaceutically acceptable carrier.
 15. The pharmaceutical compositionof claim 14, wherein the first XTEN comprises an amino acid sequencewhich has at least 90% sequence identity to a sequence selected from thegroup consisting of AE144 (SEQ ID NO: 41), AE288 (SEQ ID NO: 47), AE576(SEQ ID NO: 54) and AE864 (SEQ ID NO: 59).
 16. A kit comprising:packaging material; at least a first container comprising thepharmaceutical composition of claim 14; a label identifying thepharmaceutical composition and providing storage and handlingconditions; and a sheet of instructions for the reconstitution and/oradministration of the pharmaceutical composition to a subject.
 17. Amethod of treating a human disease, the method comprising administeringa therapeutically effective dose of the pharmaceutical composition ofclaim 14 to direct cytotoxic activity of T-cells towards tumor cells.18. The method of claim 17, wherein the human disease is selected fromthe group consisting of breast carcinoma, lung carcinoma, gastriccarcinoma, esophageal carcinoma, colorectal carcinoma, liver carcinoma,ovarian carcinoma, thecoma, arrhenoblastoma, cervical carcinoma,endometrial carcinoma, fibrosarcoma, choriocarcinoma, head and neckcancer, nasopharyngeal carcinoma, laryngeal carcinoma, hepatoblastoma,Kaposi's sarcoma, melanoma, skin carcinoma, hemangioma, cavernoushemangioma, hemangioblastoma, pancreas carcinoma, retinoblastoma,astrocytoma, glioblastoma, Schwannoma, oligodendroglioma,medulloblastoma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma,leiomyosarcomas, urinary tract carcinoma, thyroid carcinoma, Wilm'stumor, renal cell carcinoma, and prostate carcinoma.
 19. The isolatedbinding fusion protein of claim 7, wherein the second targeting moietycomprises a second single-chain variable fragment (scFv).